CN110945076A - Transmembrane pH gradient polymersome for quantifying ammonia in body fluids - Google Patents

Transmembrane pH gradient polymersome for quantifying ammonia in body fluids Download PDF

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CN110945076A
CN110945076A CN201880045389.8A CN201880045389A CN110945076A CN 110945076 A CN110945076 A CN 110945076A CN 201880045389 A CN201880045389 A CN 201880045389A CN 110945076 A CN110945076 A CN 110945076A
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ammonia
polymersome
sample
dye
ratio
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CN110945076B (en
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J－C·勒鲁
S·马图里
O·V·维特格尔
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Eidgenoessische Technische Hochschule Zurich ETHZ
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Abstract

The present invention provides polymersomes comprising amphiphilic block copolymers and their use for quantifying ammonia in a sample (e.g., a bodily fluid sample). More specifically, the present invention provides a polymersome comprising (a) a membrane comprising a block copolymer of poly (styrene) (PS) and poly (ethylene oxide) (PEO), wherein the PS/PEO molecular weight ratio is higher than 1.0 and lower than 4.0; and (b) a core encapsulating the acid and at least one pH-sensitive dye. Also provided are compositions, test strips and kits comprising the polymersomes and methods of using the polymersomes, compositions and kits to quantitate ammonia in a sample.

Description

Transmembrane pH gradient polymersome for quantifying ammonia in body fluids
Technical Field
The present invention relates to compositions and uses of transmembrane pH gradient polymersomes for quantifying ammonia in bodily fluids (e.g., serum, plasma, and saliva). More particularly, the invention relates to quantifying such molecules, for example, to diagnose and monitor diseases or disorders (e.g., hepatic encephalopathy).
Background
Ammonia (NH)3) Is a neurotoxic endogenous metabolite that accumulates in patients suffering from various diseases and disorders (e.g., impaired liver function (e.g., due to cirrhosis, acute liver failure, portal bypass, congenital defects in ammonia metabolism) (Matori and Leroux, Advanced Drug Delivery Reviews (ADDR) 2015; 90: 55-68)) or receiving some treatment. Ammonia can also accumulate in other environments, such as soil and wastewater.
Ammonia in body fluids
High blood ammonia levels (hyperammonemia) are associated with Hepatic Encephalopathy (HE), a serious neuropsychiatric disorder with acute and chronic manifestations that can lead to death (Vilstrup et al, Hepatology (Hepatology) 2014; 60: 715-. The prevalence of HE is high (up to 20%) in patients with cirrhosis (Vilstrup et al, supra; Blachier et al, Journal of Hepatology 2013; 58: 593-. This chronic disease usually progresses from mild (cognitive impairment) to severe (hyperammonemic coma in some patients with fatal outcome) symptoms (Vilstrup et al, supra). Plasma ammonia cutoff was 50 μ M in adults and 100 μ M in infants (Matori and Leroux, supra). In the acute hyperammonemic crisis, serum ammonia levels in excess of 1.5mM are reported (Bergmann et al, Pediatrics 2014; 133: e1072-e 1076).
Quantification of ammonia in blood or plasma is an important part of the initial diagnosis of HE and the follow-up of HE patients. HE patient's response to therapeutic intervention (e.g., lactulose therapy) is determined based in part on changes in plasma ammonia levels (Vilstrup et al, supra). In addition, plasma ammonia levels are also measured in certain drug therapies associated with hyperammonemia, such as valproic acid therapy (Vilstrup et al, supra).
Ammonia levels in semen are also associated with reduced semen quality and fertility (Kim et al, 1998).
Salivary ammonia levels are primarily affected by urease-mediated degradation of urea to ammonia in the oral cavity and thus may be a surrogate parameter for plasma urea. Since plasma urea levels are indicative of the success of hemodialysis, for example, quantifying ammonia in the oral cavity of patients with chronic kidney disease allows the caregiver to determine when the dialysis process can be terminated (Hibbard et al, analytical chemistry (Anal Chem) 2013; 85: 12158-. Because of the relatively high concentration of ammonia in saliva (about 1-8mM, Chen et al, J Breath Res 2014; 8: 036003, FIG. 5), it cannot be evaluated by established methods of ammonia quantification without prior dilution. Currently, the measurement of ammonia in many body fluids is challenging. In addition to The degradation process that requires storage of The sample at low temperatures to prevent ammonia production in The sample, available methods for ammonia quantification have significant limitations (Barsotti Journal of Pediatrics 2001; 138: S11-S20). Due to the low selectivity, the fibrate reaction (Berthelot reaction) of the indoxyl-forming reaction based on phenol, ammonia and hypochlorite (fig. 1) is strongly influenced by primary amines (e.g. amino acids, proteins), which hinders its use in biological fluids. In enzyme-based ammonia assays (e.g., Randox ammonia assay AM1015, Randox Laboratories, Schweitz, Switzerland), glutamate dehydrogenase is assayed in NAD (P) H to NA (D) P+Ammonia and α -ketoglutaric acid to L-glutamic acid and water due to NAD (P) H and NAD (P)+The absorption spectrum of (a) is different, so that the progress of the reaction can be followed spectrophotometrically, and the ammonia concentration can be determined. The upper limit of quantitation for most commercial assays based on glutamate dehydrogenase is about 1.2 mM. Unfortunately, enzymatic ammonia quantitation methods are subject to a variety of factors (e.g., lipids, heavy metals (e.g., zinc or iron),With NAD (P) H or NAD (P)+Enzymes of reaction, tannins), and because of the strong time dependence of the enzyme reaction, this method relies on precise time to produce reliable results (Seiden-Long et al, Clinical Biochemistry 2014; 47: 1116-1120). This complicates high throughput experiments. PocketChemTMThe BA blood ammonia analyzer is a test strip-based system for the immediate ammonia detection in capillary blood. When the sample penetrates the strip, it is alkalinized, converting the ammonium to ammonia. Subsequently, the ammonia passes through the hydrophobic membrane and causes a change in pH on the indicator strip, which is quantified spectrophotometrically. PocketChem due to its negative constants and proportional bias, low throughput (3 minutes per measurement) and low upper limit of quantitation (0.285mM)TMBA ammonia meter PA-4140 has not been widely used preclinical and clinical (Goggs et al, Veterinary clinical Pathology (Veteriary clinical Pathology) 2008; 37: 198-.
Other samples
Ammonia is a common contaminant of soil and water (due to, for example, ammonia-containing fertilizers or industrial waste (Mook et al, Desalination 2012; 285: 1-13) — it is typically quantified using ammonium ion-selective electrodes (Mook et al, supra).
There is a need for alternative quantification tools for ammonia in samples, including body fluids.
This specification is directed to a number of documents, the entire contents of which are incorporated herein by reference in their entirety.
Disclosure of Invention
The present invention describes compositions and uses of transmembrane pH gradient polymersomes for quantifying (e.g., determining the concentration of) ammonia in body fluids. It uses, for example, polymersomes composed of amphiphilic block copolymers (e.g., poly (styrene) -b-poly (ethylene oxide) (PS-b-PEO, also known as poly (styrene) -b-poly (ethylene glycol), PS-b-PEG)).
Using pH sensitive dyes (e.g., fluorescein (trisodium 8-hydroxypyrene-1, 3, 6-trisulfonate, i.e., HPTS trisodium salt),LysosensorTMYellow/blue dextran conjugate, 8-aminonaphthalene-1, 3, 6-trisulfonic acid disodium salt (ANTS), IRDyeTM680RD carboxylate, etc.) to quantify the pH rise in the polymer vesicle core due to ammonia capture.
Polymersomes composed of amphiphilic block copolymer poly (styrene) -b-poly (ethylene glycol) (PS-b-PEO) and an acidic component (see e.g. co-pending PCT application PCT/IB2017/054966 filed on 8, 15, 2017) were used to measure the concentration of ammonia in body fluids. For the preparation of polymersomes, organic solvents are used. The polymer is dissolved in a suitable solvent (e.g., dichloromethane) and emulsified in an acidic solution (e.g., citric acid) or sodium chloride solution containing a pH-sensitive dye. After removal of the solvent, the polymersomes are purified to remove unencapsulated dye (if any) and other weak acids. Then, upon exposure to neutral or high pH solutions, the polymersomes exhibit absorption properties for ammonia. The efficacy of polymersomes was demonstrated by showing quantification of ammonia in buffer as well as in native and spiked serum, plasma, saliva, urine, sweat and semen (figures 3-5 and 7-9).
The polymersomes of the invention may be used for the quantification of ammonia (or indirectly, other biomarkers that can be converted in vitro to a corresponding (1-fold or more) amount of ammonia (e.g., an amino acid such as phenylalanine)). In more specific embodiments, the polymersomes of the invention are useful in the assay of bodily fluids to diagnose ammonia-related diseases or disorders (e.g., hyperammonemia), to follow-up patients with ammonia-related diseases or disorders, and for research and preclinical use. The diagnostic products of the invention are useful for ammonia measurement in vitro, preclinical (e.g., animal studies) and clinical studies, as well as in routine clinical practice, where ammonia quantification is required. Ammonia measurements can be used for diagnosis and staging of ammonia-related diseases or conditions, as well as for assessing the response of hyperammonemic patients to treatment for hyperammonemia or the response of patients at risk of hyperammonemia to preventive measures. The invention may further be used to quantify ammonia in vitro assays, more specifically to identify compounds that inhibit ammonia production.
The transmembrane pH-gradient PS-b-PEO polymersomes of the invention may advantageously show high selectivity for ammonia (fig. 6) (presumably, but not limited by this hypothesis, due to the impermeability of highly hydrophobic polystyrene membranes) and a large detection range of at least about 0.005mM to about 8mM (fig. 2 and 13). A high upper limit of quantitation would be advantageous as it would at least reduce (even eliminate) the need to dilute the concentrated ammonia sample.
Furthermore, the kinetics of ammonia uptake into polymersomes was fast and time independent (low time dependence) after 2.5 min incubation at physiological pH (fig. 2). These features enable analysis of a large number of samples simultaneously.
More specifically, according to one aspect of the present invention, the following subject matter is provided:
a polymersome comprising (a) a membrane comprising a block copolymer of poly (styrene) (PS) and poly (ethylene oxide) (PEO), wherein the PS/PEO molecular weight ratio is higher than 1.0 and lower than 4.0; and (b) a core encapsulating the acid and at least one pH-sensitive dye.
The polymersome according to item 1, wherein the block copolymer is a diblock copolymer.
Polymersomes according to item 1 or 2, wherein the concentration of the acid is such that when the polymersome is hydrated, a pH between 1 and 6.5, 2 and 6, 2 and 5.5 or 3 and 5.5 results.
The polymersome according to any one of claims 1 to 3, wherein the acid is in an acidic aqueous solution.
The polymersome of item 5. the polymersome of item 4, wherein the pH in the acidic aqueous solution is between 1 and 6.5, 2 and 5.5, or 3 and 5.5.
The polymersome of any one of claims 1 to 5, wherein the at least one pH-sensitive dye comprises (i) hydroxypyrene; (ii) phenyl pyridyl oxazole; (iii) an aminonaphthalene; (iv) a cyanine; or (v) any pH-sensitive fluorescent derivative of any one of (i) to (iv).
Item 7 the polymersome of item 6, wherein the pH sensitive dye comprises 8-hydroxypyrene-1, 3, 6-trisulfonate (HPTS), dextran conjugated LysosensorTMYellow/blue, 8-aminonaphthalenes-1,3, 6-trisulfonate (ANTS) or IRDyeTM680RD carboxylate.
The polymersome of any one of claims 1 to 7, wherein the acid and the at least one pH-sensitive dye are different molecules.
The polymersome of any one of claims 1 to 8, wherein the acid is a hydroxy acid, most preferably citric acid.
The polymersome of any one of claims 1 to 7, wherein the acid and the at least one pH-sensitive dye are the same molecule.
The polymersome according to any one of claims 1 to 10, which is prepared by a method comprising mixing an organic solvent containing the copolymer with an aqueous phase containing the acid and at least one pH-sensitive dye.
The polymersome of claim 12, the polymer vesicle of claim 11, wherein the organic solvent is water insoluble or partially water soluble.
The polymersome according to any one of claims 1 to 12, wherein the pH-sensitive dye is a pH-sensitive fluorescent dye.
The polymersome according to any one of claims 1 to 12, wherein the pH-sensitive dye is a pH-sensitive absorbing dye.
A method of preparing the polymersome of any one of claims 1 to 14, comprising:
(a) dissolving the block copolymer of PS and PEO in an organic solvent (preferably a water-insoluble or partially water-soluble organic solvent) to form a copolymer-containing organic phase;
(b) mixing said organic solvent phase containing copolymer with an aqueous phase containing acid and at least one pH sensitive dye to form said polymersome; and
(c) removing the at least one pH sensitive dye and the organic solvent that are not encapsulated.
The process of claim 15, wherein the aqueous phase comprises 0.2 to 100mM of an acid.
Item 17. a polymersome prepared according to the method described in item 15 or 16.
The polymersome of any one of items 1 to 14 and 17, wherein a core of the polymersome further encapsulates ammonia.
A composition comprising the polymersome of any one of items 1 to 14 and 17 and at least one excipient.
The composition of claim 19, wherein the at least one excipient comprises a preservative, a cryoprotectant, a lyoprotectant, an antioxidant, or a combination of at least two thereof.
The composition of item 21. the composition of item 19 or 20, wherein the composition is in liquid or solid form.
A test strip comprising the composition in solid form according to item 19 or 20.
Item 23. the polymersome according to any one of items 1 to 14 and 17, or the composition according to any one of items 19 to 21, or the strip according to item 22, for quantification of ammonia in a fluid sample.
The polymersome, composition, or strip for use according to item 23, wherein the sample comprises a bodily fluid from a subject.
The polymersome, the composition for use according to item 24, wherein the sample further comprises a buffer.
The polymersome, composition, or strip for use according to item 24, wherein the subject (i) has an ammonia-related disease or disorder or phenylketonuria; (ii) suspected or likely to suffer from an ammonia-related disease or disorder or phenylketonuria; or (iii) is undergoing treatment for anti-hyperammonemia or anti-phenylketonuria.
A method of determining the concentration of ammonia in a sample using the polymersome of any one of items 1 to 14 and 17, the composition of any one of items 19 to 21, or the test strip of item 22, comprising:
(a) contacting the polymersome, composition, or strip with the sample;
(b) determining at least one pH-dependent spectral characteristic in the sample containing polymeric vesicles or composition or the test strip containing the sample; and
(c) determining the concentration of ammonia in the sample using the at least one pH-dependent spectral characteristic by reference to a standard curve.
Item 28 the method of item 27, wherein the pH-dependent spectral characteristic is pH-dependent absorbance, the pH-sensitive dye is a pH-dependent absorbing dye, and the standard curve is an absorbance standard curve.
The method of item 29, wherein the pH-dependent spectral characteristic is pH-dependent fluorescence intensity, the pH-sensitive dye is a pH-sensitive fluorescent dye, and the standard curve is a fluorescent standard curve.
The method of item 27, wherein (b) further comprises measuring at least one pH independent spectral characteristic or at least one additional pH dependent spectral characteristic in the sample containing the polymer vesicles or composition or the test strip containing the sample to calculate at least one spectral characteristic ratio, and wherein (c) the ammonia concentration in the sample containing the polymer vesicles or composition or the test strip containing the sample is determined by reference to a spectral characteristic ratio standard curve using the at least one pH dependent spectral characteristic ratio.
The method of item 31, item 30, wherein the at least one pH-dependent spectral characteristic and the at least one pH-independent spectral characteristic are produced by the same pH-sensitive dye.
Item 32 the method of item 30 or 31, wherein the spectral characteristic is absorbance and the pH-sensitive dye is a pH-sensitive absorbing dye.
The method of items 30 or 31, wherein the spectral characteristic is fluorescence and the pH-sensitive dye is a pH-sensitive fluorescent dye.
The method of any of claims 27-33, wherein the pH in the polymeric vesicle core is between 2 and 6.5.
The method of any of claims 27-34, wherein the at least one pH-sensitive dye comprises hydroxypyrene or one of its derivatives.
Item 36 the method of item 35, wherein the at least one pH-sensitive dye comprises 8-hydroxypyrene-1, 3, 6-trisulfonate (HPTS).
The method of any one of claims 27 to 34, wherein the at least one pH-sensitive dye comprises pyridylphenyloxazole or one of its derivatives; aminonaphthalene or one of its derivatives; or a cyanine or one of its derivatives.
The method of item 38, wherein the at least one pH-sensitive dye comprises a dextran-conjugated LysosensorTMYellow/blue, ANTS or IRDyeTM680RD carboxylate.
The method of any one of claims 27-38, wherein the sample comprises a sample of bodily fluid from a subject.
Item 40 the method of item 39, wherein the bodily fluid is a blood or blood fraction sample, a saliva sample, or a semen sample.
The method of item 41, wherein the body fluid has been pretreated with phenylalanine ammonia lyase.
Item 42. the method of item 40 or 41 for (i) diagnosing an ammonia-related disease or disorder or phenylketonuria in the subject, wherein an ammonia concentration in the sample that is higher than a reference ammonia concentration is indicative of the subject having an ammonia-related disease or disorder or phenylketonuria; or for (ii) monitoring the effectiveness of an anti-hyperammonemia or anti-phenylketonuria treatment, wherein an ammonia concentration in the sample that is lower than a reference ammonia concentration indicates that the anti-hyperammonemia or anti-phenylketonuria treatment is effective.
A kit for determining the ammonia concentration in a sample comprising (a) a polymersome according to any one of items 1 to 14 and 17, a composition according to any one of items 19 to 21 or a strip according to item 22, and (b) (i) a solution for hydrating the polymersome; (ii) a buffer for adjusting the outer phase of the polymersome and/or the pH of the sample to be tested; (iii) a diluent for diluting a sample to be tested; (iv) a fluorescence standard curve and/or an absorbance standard curve; (v) one or more solutions of known ammonia concentration; or (vi) a combination of at least two of (i) to (v).
Other objects, advantages and features of the present invention will become more apparent upon reading of the following non-restrictive description of specific embodiments thereof, given by way of example only with reference to the accompanying drawings.
Drawings
In the drawings:
FIG. 1 shows the effect of L-lysine on ammonia quantification based on the fibulo reaction. The presence of L-lysine leads to an insufficient estimate of ammonia concentration using the fibulo reaction. The results are expressed as mean and standard deviation (n-3).
FIG. 2 shows the fluorescence intensity ratio of PS-b-PEO transmembrane pH-gradient polymersome containing a fluorescent yellow dye in phosphate buffer at different ammonia concentrations. The fluorescence intensity ratio of the PS-b-PEO polymersomes containing the fluorescent yellow dye is a function of the ammonia concentration in the medium. The results are expressed as mean and standard deviation (n-3).
Figure 3 compares the quantification of ammonia in human serum by fluorescent PS-b-PEO polymersomes and by a commercial enzymatic ammonia assay. Similar to the enzyme kit, transmembrane pH-gradient PS-b-PEO polymersomes containing fluorescent yellow dye were able to quantify ammonia in native and spiked human serum. Results are expressed as mean and standard deviation (n-3 for polymersome assay; n-8 for enzyme kit).
Figure 4 compares the quantification of ammonia in human plasma by fluorescent PS-b-PEO polymersomes and by a commercial enzymatic ammonia assay. Similar to the enzyme kit, transmembrane pH-gradient PS-b-PEO polymersomes containing fluorescent yellow dye were able to quantify ammonia in native and spiked human plasma. The results are expressed as mean and standard deviation (n-3).
Figure 5 compares the quantification of ammonia in human saliva by fluorescent PS-b-PEO polymersomes and by a commercial enzymatic ammonia assay. Similar to the enzyme kit, transmembrane pH-gradient PS-b-PEO polymersomes containing fluorescent yellow dye were able to quantify ammonia in natural and spiked human saliva. The results are expressed as mean and standard deviation (n-3).
FIG. 6 shows the effect of L-lysine on ammonia quantification based on PS-b-PEO polymersomes. The presence of L-lysine up to 15mM (i.e., 100 times the normal plasma concentration) did not affect the ammonia concentration measured. The results are expressed as mean and standard deviation (n-3).
Figure 7 compares the quantification of ammonia in human urine by fluorescent PS-b-PEO polymersomes and by a commercial enzymatic ammonia assay. Similar to the enzyme kit, transmembrane pH-gradient PS-b-PEO polymersome containing a fluorescent yellow dye was able to quantify ammonia in natural and spiked human urine. The results are expressed as mean and standard deviation (n-3).
Figure 8 compares the quantification of ammonia in human sweat by fluorescent PS-b-PEO polymer vesicles and by a commercial enzymatic ammonia assay. Similar to the enzyme kit, transmembrane pH gradient PS-b-PEO polymersomes containing fluorescent yellow dye were able to quantify ammonia in native and spiked human sweat. The results are expressed as mean and standard deviation (n-3).
Figure 9 compares the quantification of ammonia in human seminal fluid by fluorescent PS-b-PEO polymersomes and by a commercial enzymatic ammonia assay. Similar to the enzyme kit, transmembrane pH-gradient PS-b-PEO polymersomes containing fluorescent yellow dye were able to quantify ammonia in natural and spiked human semen. The results are expressed as mean and standard deviation (n-3).
FIG. 10 shows dextran conjugate containing Lysosensor in phosphate buffer at different ammonia concentrationsTMFluorescence intensity ratio of yellow/blue PS-b-PEO polymersomes. Dextran-conjugated LysosensorTMThe fluorescence intensity ratio of the yellow/blue PS-b-PEO polymersomes is a function of the ammonia concentration in the medium. The results are expressed as mean and standard deviation (n-3).
FIG. 11 shows the fluorescence intensity ratio of ANTS-containing PS-b-PEO polymersomes at different ammonia concentrations in phosphate buffer. The fluorescence intensity ratio of ANTS-containing PS-b-PEO polymersomes is a function of the ammonia concentration in the medium. The results are expressed as mean and standard deviation (n-3).
FIG. 12 shows IRDye-containing solutions in phosphate buffer at different ammonia concentrationsTM680RD of PS-b-PEO polymersome. Containing IRDyeTMThe fluorescence intensity of the 680RD PS-b-PEO polymersome is a function of the ammonia concentration in the medium. The results are expressed as mean and standard deviation (n-3).
FIG. 13 shows the fluorescence intensity ratio of PS-b-PEO polymersomes containing fluorescent yellow dye in phosphate buffer at different ammonia concentrations, with no additional acid present in the polymer vesicle core. The fluorescence intensity ratio of the PS-b-PEO polymersomes containing the fluorescent yellow dye is a function of the ammonia concentration in the medium in the absence of other acids in the polymer vesicle core. The results are expressed as mean and standard deviation (n-3).
FIG. 14 shows the quantification of ammonia by fluorescent yellow dye-containing PS-b-PEO polymersomes (PS/PEO ratio of about 1.2 and about 3) in phosphate buffer. Transmembrane pH gradient PS-b-PEO polymersome containing a fluorescent yellow dye (PS/PEO ratio of about 1.2 and about 3.0) was able to quantify ammonia in phosphate buffer. The results are expressed as mean and standard deviation (n-3).
FIG. 15 shows the absorbance ratio of PS-b-PEO polymer vesicles containing fluorescent yellow dye at different ammonia concentrations in phosphate buffer. The results are expressed as mean and standard deviation (n-3).
Detailed Description
The invention includes transmembrane pH gradient polymersomes for quantifying ammonia in bodily fluids, compositions comprising the polymersomes, methods of making the polymersomes, and uses of the polymersomes and compositions.
Polymersome
A polymersome is a vesicle whose bilayer membrane is assembled from synthetic copolymers. The average diameter is 50nm to 100 μm or more, and in one embodiment, 100nm to 40 μm, as determined by laser diffraction. Although the test polymersomes of the present invention having average diameters varying between 100nm and 40 μm were able to effectively encapsulate ammonia, there is no reason to believe that polymersomes having diameters greater than 40 μm are ineffective.
The polymersome of the present invention comprises an amphiphilic block copolymer and is prepared using an organic solvent.
The mechanism of action of the present invention is based on a pH gradient across the polymer vesicle membrane. In various fluid samples (e.g., bodily fluid samples) used in the present invention, the pH of the acidic reagent contained in the aqueous polymer vesicle core is different from (lower than) the pH of the sample (e.g., physiological pH) or the pH of the sample after addition of a buffer. Typically, the pH of human blood and its liquid fractions is between 7.35 and 7.45; the pH of saliva is between 6.7 and 7.4 (Baliga et al, J Indian Soc Periodontotol 2013, 17: 461-; the pH of urine is between 4.5 and 7.5 (Malouf et al, Journal of the American Society of Nephrology 2007 (Clinical Journal of the American Society of neurology), 2: 883-; the pH of sweat is between 4.5 and 7 (Oncecu et al, 2013 in Lab Chip; 13: 3232-3238); semen has a pH of between 7.2 and 8.0 (Haugen et al, J. International journal of Male sciences 1998; 21: 105-. These ranges are similar in other mammals. Thus, ammonia can diffuse through the hydrophobic polymeric membrane of the polymersome in its uncharged state and then be trapped in the internal compartment in its protonated (ionized) state (e.g., where the ammonia is ammonium). Although ammonia is present primarily in its protonated state at the pH of the sample (e.g., body fluid), used directly or diluted with a buffer, there will always be a small portion of ammonia in the non-ionized state. This moiety can diffuse within the polymersome and be trapped within the polymersome in its protonated state. Protonation of the ammonia molecule consumes protons, thereby increasing the pH in the polymer vesicle core. The ammonia/ammonium equilibrium is quickly reestablished in the external phase (i.e., the ammonium deprotonates to reestablish the ammonia moiety) producing more ammonia molecules that can diffuse into the polymer vesicle core. The spectral characteristics (fluorescence intensity or absorbance) of pH-sensitive dyes within the core of polymersomes at pH-dependent wavelengths change with increasing ammonia concentration in the core.
As used herein, the property of a "transmembrane pH gradient to quantify ammonia" refers to the ability of the polymersome of the present invention to sequester ammonia when diluted in a sample (e.g., a bodily fluid sample), which may itself be diluted in a buffer.
Block copolymer
A "polymer" is a macromolecule comprising linked monomer units. The monomer units may be of a single type (homopolymer) or of multiple types (copolymer). Copolymers made from a series of two or more monomers of a single type (block) covalently linked to two or more monomers of another type (another block) are referred to as block copolymers. Copolymers composed of two block types covalently linked together are called diblocks, copolymers composed of three block types are called triblocks, and so on. As a result of the particular synthesis used to produce the block copolymer, the block copolymer may contain different end groups.
The polymersome of the present invention comprises a block copolymer. In a specific embodiment, the block copolymer of the present invention is a diblock copolymer or a triblock copolymer. These block copolymers are amphiphilic and are formed from at least two polymers, namely an aromatic highly hydrophobic polymer (e.g., poly (styrene)) and a hydrophilic uncharged and non-biodegradable polymer. In more specific embodiments, the block copolymer is a diblock copolymer (e.g., poly (styrene) -b-poly (ethylene oxide) (PS-b-PEO)) or a triblock copolymer (e.g., PEO-b-PS-b-PEO)) (i.e., a PS PEO block copolymer).
An "amphiphilic" copolymer is a copolymer containing hydrophilic (water-soluble) and hydrophobic (water-insoluble) groups.
As used herein, the term "non-biodegradable" means non-hydrolyzable (e.g., resistant to degradation by pH, enzymes, or other means) in a fluid sample condition (e.g., a bodily fluid sample).
Hydrophobic uncharged polymers
In one embodiment, the hydrophobic uncharged polymer used in the copolymers of the present invention is poly (ethylene) (CH)2-CH(C2H5))n-, i.e. - (C)4H8)n-) or poly (styrene) ((CH)2-CH(Ph))n-, i.e. - (CH)2-CH(C6H5))n-or- (C)8H8)n-). In a specific embodiment, the hydrophobic uncharged polymer is poly (styrene) (PS). The poly (styrene) s used in the present invention may comprise unsubstituted and/or substituted/functionalized styrene monomers. Thus, unless otherwise specifically defined, the term "poly (styrene)" is used herein to generally refer to a poly (styrene) comprising fully unsubstituted styrene monomers, mixtures of substituted and unsubstituted styrene monomers, or fully substituted styrene monomers. The one or more substituents on the styrene monomer may include substituents on the phenyl group and/or on the carbon to which the phenyl group is attached, and/or may form a polycyclic derivative with the phenyl group (e.g., bicyclic, tricyclic, etc., containing C3-C6 aryl and/or C3-C6 cycloalkyl). Possible substituents include alkyl (C1-C7(C1, C2, C3, C4, C5, C6, or C7, more specifically, C1, C2, or C3)), aryl (C3-C6), C3-C8 cycloalkyl, arylalkyl, acetoxy, alkoxy (methoxy, ethoxy, propoxy, butoxy, etc.), halogen (Br, Cl, F, etc.), amine, amide, alkylamine, NO2The substituent itself may be substituted, without limitation, and substituted styrene monomers include acetoxystyrene, benzhydrylstyrene, benzyloxymethoxystyrene, bromostyrene (2-, 3-, 4-or α), chlorostyrene (2-, 3-, 4-or α), fluorostyrene (2-, 3-, 4-or α), t-butoxystyrene, t-butylstyrene, chloromethylstyrene, dichlorostyrene, difluorostyrene, dimethoxystyrene, dimethylstyrene, dimethylvinylbenzylamine, diphenylmethylpentene, (diphenylphosphino) styrene, ethoxystyrene, isopropenylaniline, isopropenyl- α -dimethylbenzyl isocyanate, [ N- (methylaminoethyl) aminomethyl]Styrene, methylstyrene, nitrostyrene, 4-vinylbenzoic acid pentafluorophenyl ester, pentafluorostyrene, (trifluoromethyl) styrene (2-, 3-or 4-), trimethylstyrene, vinylaniline (3-or 4-), methoxystyrene, vinylbenzoic acid (3-, 4-), vinylbenzyl chloride, (vinylbenzyl) trimethylammonium vinylbi-partBenzene, 4-vinylbenzocyclobutene (4-, etc.), vinylanthracene (9-, etc.), 2-vinylnaphthalene, vinylbiphenyl (3-, 4-, etc.), and the like. In one embodiment, the PS comprises at least one substituted styrene monomer. The substituent may be a nonionic group (e.g., methyl or t-butyl). In particular embodiments, the substituted styrene monomer is an alkylstyrene (e.g., methylstyrene) or t-butylstyrene. In another embodiment, the styrene monomer in the poly (styrene) is unsubstituted.
Hydrophilic uncharged polymers
Hydrophilic uncharged polymers that can be used with the poly (styrene) in the block copolymers of the present invention include polymers of poly (ethylene oxide), poly (vinyl pyrrolidone), poly (ethyloxazoline), poly (methyloxazoline), and oligoethylene glycol alkyl acrylates. In a particular embodiment, the hydrophilic uncharged polymer is poly (ethylene oxide).
The poly (ethylene oxide) (PEO) used in the present invention has the general formula: (O-CH)2-CH2)n-, i.e. - (C)2H4O)n-) and includes unsubstituted and substituted/functionalized ethylene oxide monomers. Thus, unless otherwise specifically defined, the term "poly (ethylene oxide)" or "PEO" is used herein to generally mean a composition comprising a fully unsubstituted ethylene oxide monomer, a mixture of substituted and unsubstituted ethylene oxide monomers, or a fully substituted ethylene oxide monomer. In one embodiment, the PEO comprises at least one substituted ethylene oxide monomer. In another embodiment, the ethylene oxide monomers are unsubstituted.
Proportion of Polymer
The molecular weight of the PS and PEO blocks (e.g., diblock PS-b-PEO or triblock PEO-b-PS-b-PEO) can vary so long as the bilayer structure and stability is maintained. The present inventors found that stable PS-b-PEO polymersomes were formed between PS/PEO number average molecular weight ratios above 1.0 and below 4 (see, e.g., examples 2-16). In one embodiment, the ratio is about 1.1 or more and less than 4. In another embodiment, the ratio is about 1.2 or more and less than 4. In another embodiment, the ratio is about 1.3 or more and less than 4. In another embodiment, the ratio is about 1.4 or more and less than 4. In another embodiment, the ratio is greater than 1 and about 3.9 or less. In one embodiment, the ratio is about 1.1 or more and less than 3.9. In another embodiment, the ratio is about 1.2 or more and less than 3.9. In another embodiment, the ratio is about 1.3 or more and less than 3.9. In another embodiment, the ratio is about 1.4 or more and less than 3.9. In another embodiment, the ratio is greater than 1 and about 3.8 or less. In one embodiment, the ratio is about 1.1 or more and less than 3.8. In another embodiment, the ratio is about 1.2 or more and less than 3.8. In another embodiment, the ratio is about 1.3 or more and less than 3.8. In another embodiment, the ratio is about 1.4 or more and less than 3.8. In another embodiment, the ratio is greater than 1 and about 3.7 or less. In one embodiment, the ratio is about 1.1 or more and less than 3.7. In another embodiment, the ratio is about 1.2 or more and less than 3.7. In another embodiment, the ratio is about 1.3 or more and less than 3.7. In another embodiment, the ratio is about 1.4 or more and less than 3.7. In another embodiment, the ratio is greater than 1 and about 3.6 or less. In one embodiment, the ratio is about 1.1 or more and less than 3.6. In another embodiment, the ratio is about 1.2 or more and less than 3.6. In another embodiment, the ratio is about 1.3 or more and less than 3.6. In another embodiment, the ratio is about 1.4 or more and less than 3.6. In another embodiment, the ratio is greater than 1 and about 3.5 or less. In one embodiment, the ratio is about 1.1 or more and less than 3.5. In another embodiment, the ratio is about 1.2 or more and less than 3.5. In another embodiment, the ratio is about 1.3 or more and less than 3.5. In another embodiment, the ratio is about 1.4 or more and less than 3.5. In another embodiment, the ratio is greater than 1 and about 3.4 or less. In one embodiment, the ratio is about 1.1 or more and less than 3.4. In another embodiment, the ratio is about 1.2 or more and less than 3.4. In another embodiment, the ratio is about 1.3 or more and less than 3.4. In another embodiment, the ratio is about 1.4 or more and less than 3.4. In another embodiment, the ratio is greater than 1 and about 3.3 or less. In a particular embodiment, the ratio is about 1.1 or more and less than 3.3. In another embodiment, the ratio is about 1.2 or more and less than 3.3. In another embodiment, the ratio is about 1.3 or more and less than 3.3. In another embodiment, the ratio is about 1.4 or more and less than 3.3. In another embodiment, the ratio is greater than 1 and about 3.2 or less. In one embodiment, the ratio is about 1.1 or more and less than 3.2. In another embodiment, the ratio is about 1.2 or more and less than 3.2. In another embodiment, the ratio is about 1.3 or more and less than 3.2. In another embodiment, the ratio is about 1.4 or more and less than 3.2. In another embodiment, the ratio is greater than 1 and about 3.2 or less. In one embodiment, the ratio is about 1.1 or more and less than 3.1. In another embodiment, the ratio is about 1.2 or more and less than 3.1. In another embodiment, the ratio is about 1.3 or more and less than 3.1. In another embodiment, the ratio is about 1.4 or more and less than 3.1. In one embodiment, the ratio is about 1.1 or more and less than 3. In another embodiment, the ratio is about 1.2 or more and less than 3. In another embodiment, the ratio is about 1.3 or more and less than 3. In another embodiment, the ratio is about 1.4 or more and less than 3. In another embodiment, the ratio is greater than 1 and about 2.9 or less. In another embodiment, the ratio is about 1.1 or more and about 2.9 or less. In another embodiment, the ratio is about 1.2 or more and about 2.9 or less. In another embodiment, the ratio is about 1.3 or more and about 2.9 or less. In another embodiment, the ratio is about 1.4 or more and about 2.9 or less. In another embodiment, the ratio is greater than 1 and about 2.8 or less. In another embodiment, the ratio is about 1.1 or more and about 2.8 or less. In another embodiment, the ratio is about 1.2 or more and about 2.8 or less. In another embodiment, the ratio is about 1.3 or more and about 2.8 or less. In another embodiment, the ratio is about 1.4 or more and about 2.8 or less. In another embodiment, the ratio is greater than 1 and about 2.7 or less. In another embodiment, the ratio is greater than about 1.1 and about 2.7 or less. In one embodiment, the ratio is about 1.2 to about 2.7 or less. In one embodiment, the ratio is about 1.3 or more and about 2.7 or less. In one embodiment, the ratio is about 1.4 or more and about 2.7 or less. In another embodiment, the ratio is greater than 1 and about 2.6 or less. In another embodiment, the ratio is greater than about 1.1 and about 2.6 or less. In one embodiment, the ratio is about 1.2 to about 2.6 or less. In one embodiment, the ratio is about 1.3 or more and about 2.6 or less. In one embodiment, the ratio is about 1.4 or more and about 2.6 or less. In another embodiment, the ratio is greater than 1 and about 2.5 or less. In another embodiment, the ratio is greater than about 1.1 to about 2.5 or less. In one embodiment, the ratio is about 1.2 to about 2.5 or less. In one embodiment, the ratio is about 1.3 or more and about 2.5 or less. In one embodiment, the ratio is about 1.4 or more and about 2.5 or less. In another embodiment, the ratio is greater than 1 and about 2.4 or less. In another embodiment, the ratio is greater than about 1.1 to about 2.4 or less. In one embodiment, the ratio is about 1.2 to about 2.4 or less. In one embodiment, the ratio is about 1.3 or more and about 2.4 or less. In one embodiment, the ratio is about 1.4 or more and about 2.4 or less. In another embodiment, the ratio is greater than 1 and about 2.3 or less. In another embodiment, the ratio is greater than about 1.1 and about 2.3 or less. In one embodiment, the ratio is about 1.2 to about 2.3 or less. In one embodiment, the ratio is about 1.3 or more and about 2.3 or less. In one embodiment, the ratio is about 1.4 or more and about 2.3 or less. In another embodiment, the ratio is greater than 1 and about 2.2 or less. In another embodiment, the ratio is greater than about 1.1 to about 2.2 or less. In one embodiment, the ratio is about 1.2 to about 2.2 or less. In one embodiment, the ratio is about 1.3 or more and about 2.2 or less. In one embodiment, the ratio is about 1.4 or more and about 2.2 or less. In another embodiment, the ratio is greater than 1 and about 2.1 or less. In another embodiment, the ratio is greater than about 1.1 to about 2.1 or less. In one embodiment, the ratio is about 1.2 to about 2.1 or less. In one embodiment, the ratio is about 1.3 or more and about 2.1 or less. In one embodiment, the ratio is about 1.4 or more and about 2.1 or less. In another embodiment, the ratio is greater than 1 and about 2.0 or less. In another embodiment, the ratio is greater than about 1.1 and about 2.0 or less. In one embodiment, the ratio is about 1.2 to about 2.0 or less. In one embodiment, the ratio is about 1.3 or more and about 2.0 or less. In one embodiment, the ratio is about 1.4 or more and about 2.0 or less.
Without limiting the generality of the above statements, the present invention comprises PEO having a molecular weight of about 400g/mol to 20000 g/mol. However, applicants have no reason to expect higher molecular weight PEOs to be ineffective for use in the present invention. Polymers with lower molecular weights are easier to handle. Typically, PEO has a molecular weight of 1000 to 5000 g/mol. The molecular weight of PS is chosen to satisfy the above ratio. According to the invention, when the molecular weight of PEO is, for example, about 20000g/mol, the PS molecular weight is below about 80000 g/mol.
From hydrophobic and hydrophilic uncharged polymers (diblock copolymers or triblock copolymers) Properties of polymersomes made of substances (e.g., PS-b-PEO or PEO-b-PS-b-PEO copolymer))
Without being limited by this hypothesis, it is believed that the strong interaction of highly hydrophobic polymers in the vesicle membrane (e.g., aromatic compounds (e.g., PS) with the ability to produce pi-stacking interactions) provides for fluidic samples (e.g., body fluid samples such as serum, plasma, and saliva) and provides selectivity for ammonia (i.e., selective permeability to ammonia and poor permeability to most other biological compounds), as evidenced by the ability of the transmembrane pH gradient PS-b-PEO polymer vesicles to quantify residual ammonia in different complex biological environments and in excess primary amine L-lysine (see examples 3-9). In particular embodiments, the polymer blocks in the polymersome are non-biodegradable.
Method for producing polymersome
Preparation of the copolymer
Any known method of preparing copolymers may be used. The copolymers used in the examples described herein were purchased from Advanced Polymer Materials (Canada majority Val) (PS-b-PEO) (see examples 2-11, 14) or synthetic (see examples 12-16).
Preparation of polymersomes
The copolymer is dissolved in an organic solvent to form an organic phase and the latter is mixed with an acidic aqueous solution (e.g., a pH-sensitive dye, and optionally, if the concentration of the pH-sensitive dye is not high enough, with other acids (e.g., citric acid)) (aqueous phase). The mixing step may be performed by different techniques. For example, the polymer-containing organic phase and the aqueous phase can be mixed using an oil-in-water (o/w) emulsion, i.e., the polymer-containing organic solvent phase (i.e., the oil phase) in an acidic aqueous solution (i.e., the aqueous phase), reverse phase evaporation, nano-precipitation, or a double emulsion process.
As used herein, the term "pH-sensitive dye" refers herein to a dye whose spectral characteristics depend on the pH of the medium. In particular, it includes pH-sensitive absorbing dyes and pH-sensitive fluorescent dyes. Since the method of the present invention is based on measuring pH changes in the polymer vesicle core, the present invention encompasses all pH sensitive dyes.
As used herein, the term "absorbing dye" refers to a dye that absorbs certain ultraviolet, visible, and/or near-infrared wavelengths when illuminated by such ultraviolet, visible, and/or near-infrared wavelengths. As used herein, the term "pH-sensitive absorbing dye" refers herein to a dye whose absorption spectrum varies with pH in a medium. Without being limited thereto, the pH-sensitive absorbing dye of the present invention includes HPTS or a salt thereof (e.g., HPTS potassium salt or trisodium salt), triarylmethane dyes (e.g., bromocresol green, bromocresol purple, cresol red, chlorophenol red, phenol red, phenolphthalein, malachite green, thymol blue, bromothymol blue), azo dyes (e.g., methyl orange, methyl red, chrome black T, congo red), nitrophenol dyes (e.g., 2, 4-dinitrophenol), anthraquinone dyes (e.g., alizarin), and dyes listed below under "pH-sensitive fluorescent dyes".
The pH-sensitive absorbing dye may also comprise at least one pH-independent (isoabsorbance) wavelength, the absorbance value of which is pH-independent, and may be used to normalize the absorbance value at the pH-dependent wavelength. The pH-sensitive absorbing dye can be further normalized to the absorbance value at another pH-dependent wavelength (see example 16).
As used herein, the term "fluorescent dye" refers to a dye that produces a fluorescence intensity when irradiated at certain ultraviolet, visible, and/or near infrared wavelengths that produces or changes the fluorescence intensity of a solution in which the dye is dissolved at an appropriate concentration. As used herein, the term "pH sensitive fluorescent dye" refers to a fluorescent dye that comprises at least one pH dependent (excitation or emission) wavelength.
The pH-sensitive fluorescent dye may further comprise at least one pH-independent (absorbance) (excitation or emission) wavelength, the fluorescence intensity of which is pH-independent, and may be used to normalize the fluorescence intensity at the pH-dependent (excitation or emission) wavelength. Without being limited thereto, the pH-sensitive fluorescent dye of the present invention includes hydroxypyrene and derivatives thereof, such as HPTS or salts thereof (e.g., HPTS tripotassium or trisodium salt), phenylpyridyl oxazole and derivatives thereof, such as dextran-conjugated LysosensorTMYellow/blue, naphthalene derivatives (e.g., aminonaphthalene and its derivatives such as ANTS or its salts (e.g., disodium or dipotassium salt)), cyanines and their derivatives (e.g., IRDye)TM680RD), xanthene derivatives (e.g., fluorescein and its derivatives (e.g., carboxyfluorescein sodium), rhodamine B and its derivatives, coumarin derivatives, squaric acid derivatives, oxadiazole derivativesThe compound is selected from the group consisting of anthracene derivatives, pyrene derivatives, oxazine derivatives, acridine derivatives, arylmethine derivatives, indolinium derivatives ((E) -6-hydroxy-5-sulfo-4- (2- (1,3, 3-trimethyl-3H-indolin-1-ium-2-yl) vinyl) -2, 3-dihydro-1H-xanthene-7-sulfonate, (E) -2- (2- (6-hydroxy-7- (morpholinomethyl) -2,4 a-dihydro-1H-xanthene-3-yl) vinyl) -3, 3-dimethyl-1-propyl-3H-indol-1-ium iodide, and mixtures thereof, (E) -2- (2- (7- (benzo [ d ]))]Thiazol-2-yl) -6-hydroxy-2, 3-dihydro-1H-xanthen-4-yl) vinyl) -3, 3-dimethyl-1- (3-sulfonylpropyl) -3H-indol-1-ium-5-sulfonate) and tetrapyrrole derivatives. Notably, Near Infrared (NIR) fluorescent dyes (e.g., IRDye)TM680RD) can be used directly to assay a cell-containing body fluid sample (e.g., blood (i.e., whole blood)) (i.e., cells (e.g., red blood cells) without removing the cells from the body fluid sample prior to the fluorescence assay). Each dye has a specific pH-dependent fluorescence (excitation or emission) intensity distribution: the pH of the polymeric vesicle core is adapted to the pH range in which the dye is most sensitive to pH changes (i.e., in which the fluorescence (excitation or emission) intensity spectrum of the dye shows the strongest pH dependence). In a specific embodiment, the pH in the polymer core is between about 2 and 6.5. In a more specific embodiment, the pH in the polymer core is between about 2 and 5.5.
More than one dye may be used. For example, but not limited to, a pH sensitive fluorescent dye comprising at least one pH dependent wavelength but not comprising a pH independent wavelength may be combined with another dye comprising at least one pH independent wavelength (e.g., for calibration purposes).
The concentration of the pH-sensitive dye in the polymer vesicle core is selected to produce the appropriate absorbance or fluorescence intensity. The pH of the polymeric vesicle core and the concentration of a pH-sensitive acidic dye (e.g., a fluorescent dye) are selected to exhibit a gradual change in pH-dependent fluorescence intensity relative to the concentration of ammonia in the polymeric vesicle core in the absence of another acid. The concentration of the pH-sensitive dye is typically about 0.002 to about 200 mM. In a specific embodiment, when a pH sensitive fluorescent dye is used, the range is about 0.002 to about 200 mM. In the embodiments disclosed herein, the range is about 0.01mM (Lysosen)sorTMYellow/blue dextran, 10000MW) to about 10mM (fluorescein dye and ANTS). In the oil-in-water (o/w) emulsion process, a polymer-containing organic solvent is mixed with an aqueous acidic phase (containing a pH-sensitive dye) under ultrasound for a period of time sufficient to form an emulsion. In the following examples, the aqueous phase was saturated with organic solvent for 30 minutes with stirring before adding the polymer-containing organic solvent phase. Subsequently, the following machine-specific parameters were used: amplitude 70, cycle 0.75(UP200H, 200W, 24kHz, Hielscher sonication technique using Sonotrode S1) for 3 minutes or amplitude 10(3.1mm Sonotrode, Fisher scientific model 705 Sonic Dismembrator)TM700W, 50/60Hz, Fisher Scientific) for 2 minutes, an organic solvent phase containing the polymer was added to an aqueous phase containing the acid under sonication in an ice bath (to reduce the heat generated by the sonicator). Any method known in the art for producing an emulsion may be used. The use of sonication and the particular sonication parameters and time appropriate for preparing the emulsion will depend on the emulsion technology used. The emulsion need not be stable in the preparation process of the present invention.
In the reverse-phase evaporation method, a two-phase system comprising an organic solvent containing a polymer and a pH-sensitive dye is sonicated, and if appropriate, another aqueous phase containing an acid is sonicated to form a water-in-oil (w/o) emulsion. The outer phase was evaporated under reduced pressure until a viscous gel-like state was formed. Polymer vesicles form upon gel collapse (Krack et al, J.Am.chem.Soc.) -2008; 130: 7315-. The solvent and unencapsulated dye are subsequently removed.
In the nanoprecipitation process, the polymer is dissolved in a suitable organic solvent to which the pH-sensitive dye and, if appropriate, further acid-containing water are slowly added. Alternatively, the organic phase may be added to the aqueous phase. The solvent and unencapsulated dye are subsequently removed.
In the double emulsion process, polymersomes are formed in the form of a w/o/w double emulsion containing a pH-sensitive dye and, if appropriate, an additional acid-containing aqueous internal phase, a polymer in an intermediate phase containing a completely or partially water-insoluble organic solvent, and an aqueous external phase. The solvent and unencapsulated dye are subsequently removed.
The pH (e.g., neutral and basic pH) and composition (without buffer (i.e., dilution of the polymer vesicle-containing solution in a naturally buffered physiological solution such as serum or plasma) or with buffer) in the outer phase may also vary. Increasing the pH of the outer phase has the potential to accelerate the absorption kinetics to even higher rates due to the increased fraction of ammonia in the equilibrium ammonia/ammonium.
As used herein, "buffer" used in the outer phase of the polymersome and/or added to the body fluid sample to be tested is used to stabilize the pH or to increase the pH in order to further deprotonate the ammonium in the sample to be tested (i.e. to increase the ammonia abundance) and thereby increase the diffusion rate of ammonia in the polymersome. It is contemplated that any neutral or alkaline buffer is suitable for stabilizing the pH and any alkaline buffer is suitable for increasing the pH. Without being limited thereto, it may be more specifically a buffer containing phosphate, borate, 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid (HEPES), 3- (N-morpholinyl) propanesulfonic acid (MOPS), imidazole, bicarbonate and carbonate, or tris (hydroxymethyl) aminomethane.
The concentration of buffer in the external phase or added to the sample to be tested (e.g., body fluid) is selected to ensure the proper pH of the resulting solution, given the pH of the polymer vesicle core, to produce a sufficient pH gradient that depends on the pH profile of the dye. It is typically about 2 to about 150 mM. In one embodiment, it is about 2 to about 60mM, or about 4 to about 50 mM.
The pH of the buffer in the outer phase or of the buffer added to the sample to be tested (e.g. body fluid) is selected so as to provide a sufficient pH gradient between the outer and inner phases and to ensure a suitable kinetic profile. It is generally in the range of pH 7-10. In one embodiment, it is about pH7.4 (i.e., the pH of the PBS buffer used to prepare the ammonia standard curve) (see examples 2-16). In another embodiment, the pH of all samples to be assayed is adjusted to the same pH value as the ammonia or phenylalanine standard for comparison (see examples 2-16). In another embodiment, after addition of polymersomes having a buffered outer phase of pH7.4 to the phenylalanine sample to be determined at pH8.5 and to the phenylalanine standard curve at pH8.5, the pH of the resulting dispersion is also about 7.4 (see example 15). However, if the pH is adjusted when mixing the standard or sample with the polymersome dispersion, the pH of the sample to be tested and the standard need not be the same, but the buffering capacity of the external phase in the polymersome dispersion will set the pH and/or the pH of the resulting dispersion using additional buffer. In a particular embodiment, the pH of the outer phase of the dispersion resulting from mixing the polymersome dispersion with the sample to be tested and, if appropriate, further buffer is adjusted to be the same as the pH of the outer phase of the dispersion resulting from mixing the polymersome dispersion with the ammonia or phenylalanine standard and, if appropriate, further buffer, so that a comparison can be made. Without being limited thereto, it is also advantageous to determine the pH of 7.4 for samples of blood and blood fractions, since it corresponds to the pH of these samples, thus limiting the risk of potential pH-dependent artefacts. Any buffer added to the external phase may be omitted if a sufficient pH gradient can be created between the polymer vesicle core and the sample to be assayed by the intrinsic buffering capacity of the sample.
As used herein, the term "sufficient gradient" is generally understood to mean a difference of at least one pH unit between the core pH and the sample to be tested, and in preferred embodiments, a difference of at least 1 pH unit, preferably a difference of at least 2 pH units (e.g., pH in the sample is 6 or higher, pH in the core is 5 or lower, preferably pH in the sample is 7 or higher, pH in the core is 5 or lower). Typically, the sample to be tested inherently has or is adjusted (either by direct addition of buffer or by addition of polymersomes with a buffered outer phase) to have a pH of about 7 to about 10.
The organic solvent used in the preparation process is removed from the polymersome using any known technique. Without being limited thereto, the solvent may be removed using sub-ambient pressure applications, heating, filtration, cross-flow filtration, dialysis, or a combination of these methods.
After removal of the organic solvent, the polymersomes are purified to reduce the amount of unencapsulated pH-sensitive dye and, if necessary, to adapt the pH of the outer phase. The unencapsulated pH-sensitive dye is removed from the polymersome dispersion using any known technique. Without being limited thereto, (cross-flow) filtration, centrifugation (e.g. centrifugal filtration), size exclusion chromatography (e.g. gel permeation chromatography, gel filtration chromatography), dialysis or a combination of these methods may be employed to remove unencapsulated pH-sensitive dyes.
The purified polymersome dispersion can then be used as is (either with acidic aqueous solutions outside and inside the polymersome, or after exchange of the external acidic solution with a solution of higher pH (e.g., 7 to 10)), further dried by conventional drug drying procedures (e.g., freeze drying, spray drying), and/or incorporated into diagnostic test strips. In all these formats (as such, purified and/or dried), the polymersome core contains a pH-sensitive dye and optionally also an acid, for example if the concentration of dye is not sufficient to generate a sufficient pH gradient for the sample to be tested. When the acid and/or pH-sensitive dye is mixed with the sample (e.g., body fluid) to be tested (with or without other buffers), the acid and/or pH-sensitive dye provides a transmembrane pH gradient to the polymersome. The polymersomes of the invention so formed may also contain a salt (i.e., a partially deprotonated acid with a counter ion (such as sodium, potassium or calcium)) that may be added during the preparation of the polymersomes to adjust the pH and/or osmolarity in the core, and the polymersomes in their hydrated form also contain water. In particular embodiments, the core may also contain a preservative, which may be added during the preparation of the polymersome, to prevent microbial growth in the core, for example, in cases where the core pH is relatively high (e.g., pH 5.5 or higher). After use in the process of the invention, the core may also contain ammonia. According to particular embodiments, the contents of the polymeric vesicle core may comprise or consist of at least one pH-sensitive dye. In other embodiments, the core may further comprise at least one acid. In other embodiments, the core may further comprise at least one salt. In other embodiments, the core may further comprise water. In other embodiments, the core may further comprise at least one preservative. In other embodiments, the core may further comprise ammonia. In other embodiments, the contents of the polymeric vesicle core may consist of: (a) at least one pH sensitive dye; and (b) (i) at least one acid; (ii) at least one salt; (iii) water; (iv) at least one preservative; (v) ammonia; or (vi) a combination of at least two of (i) to (v).
When the polymersome is hydrated (i.e. contains an aqueous acidic core), the pH in its core is typically between about 1 and 6.5(1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6 or 6.5). In a particular embodiment, it is between about 1 and about 6.5, about 1 and about 6, about 1 and about 5.5, about 1 and about 4.5, about 1 and about 4, about 1.5 and about 5, about 1.5 and about 4.5, about 1.5 and about 4, about 2 and about 6.5, about 2 and about 6, about 2 and about 5.5, about 2 and about 5, about 2 and about 4.5, about 2 and about 4, about 2.5 and about 6.5, about 2.5 and about 6, about 2.5 and about 5.5, about 2.5 and about 5, about 2.5 and about 4.5, about 2.5 and about 4, about 3 and about 6.5, about 3 and about 5.5, about 3 and about 5, about 3 and about 4.5 and about 3 and about 4.5.
Although not essential to the stability of the polymersomes of the invention, the polymersome membranes may also be cross-linked. For example, Friedel-Crafts reaction with a crosslinker of p-xylylene chloride, 1, 4-bischloromethylbenzene, chlorodimethyl ether, dimethoxymethane, tris (chloromethyl) -mesitylene or p, p' -bischloromethyl-1, 4-diphenylbutane can be used to crosslink poly (styrene) (Davankov and Tsurpa, Reactive Polymers (Reactive Polymers) 1990; 13: 27-42).
Solvent(s)
The solvent used in the present invention dissolves the copolymer, and then the polymer-containing solvent is mixed with the acidic water. In the mixing step (e.g., an o/w emulsion), a fine dispersion of the polymer is formed in the aqueous phase. After the mixing step, the solvent is removed (e.g., evaporated) to ensure that the stability of the polymersome is maintained (e.g., the solvent can potentially plasticize the membrane, resulting in more permeable polymersome).
Concentrations of about 2% to about 40% (v/v) solvent phase/water phase ratio may be used. In one embodiment, the solvent phase/aqueous phase ratio is about 5% to 30% (v/v). In another embodiment, the ratio of solvent phase/aqueous phase is about 5% to 20% (v/v). In another embodiment, the ratio of solvent phase/aqueous phase is about 5% to 15% (v/v). In another embodiment, the solvent phase/aqueous phase ratio is about 10% (v/v). In a specific embodiment, the ratio of solvent phase/aqueous phase in the resulting emulsion is about 9% (v/v). In a specific embodiment, the solvent is an organic solvent.
Without being limited thereto, the solvent may be a chlorinated solvent (e.g., dichloromethane (see, e.g., examples 2-16) or chloroform), an aromatic solvent or aromatic solvent derivative (e.g., aromatic hydrocarbon or aromatic hydrocarbon derivative such as toluene), an aliphatic solvent or aliphatic solvent derivative (e.g., hexane, 1-hexanol), a ketone or ketone derivative (e.g., 2-hexanone), an ether or ether derivative (e.g., diethyl ether), or mixtures thereof (e.g., when using an o/w emulsion, a w/o/w double emulsion, or a reverse phase evaporation technique).
In one embodiment, when an o/w emulsion is used to mix the polymer-containing organic and aqueous phases, the solvent useful in the present invention is a water-insoluble or partially water-insoluble organic solvent. Without being limited thereto, such solvents include, for example, dichloromethane (see, for example, examples 2-16), chloroform, aromatic solvents or aromatic solvent derivatives (e.g., aromatic hydrocarbons or aromatic hydrocarbon derivatives such as toluene), aliphatic solvents or aliphatic solvent derivatives (e.g., hexane, 1-hexanol), ketones or ketone derivatives (e.g., 2-hexanone), ethers or ether derivatives (e.g., diethyl ether), or mixtures thereof.
Acids and acidic solutions
In particular embodiments, the pH-sensitive dye is an acid that promotes a transmembrane pH gradient. A sufficiently high concentration of a weak acid pH-sensitive dye (e.g., 10mM fluorescent yellow dye) can be used without the need for additional acid (see example 13). Without being limited thereto, the pH sensitive dye HPTS (e.g., a fluorescent yellow dye) is buffered, for example, at a pH of about 5.5 (i.e., about 1.7pH units below its pKa of 7.2) (Kano and Fendler biochem. BioPhysics 1978; 509: 289-299), which enables ammonia to be sensed in the absence of other acids. In specific examples, the acid used is not a pH sensitive dye for optimal ammonia measurement (see examples 2-12, 14-16). As used herein, the term "weak acid" refers to pKaWeak acid having a pK of 3.5 or higher andamoderately strong acids with values of-0.35 or higher (Mortimer and Mueller, Chemie, 12 th edition, Thieme, 2015).
Without being limited thereto, the acid encapsulated in the polymeric vesicle core is (i) a hydroxy acid such as citric acid, isocitric acid, malic acid, tartaric acid or lactic acid; (ii) aliphatic acids, such as short chain fatty acids (e.g., acetic acid) or unsaturated acids (e.g., sorbic acid); (iii) sugar acids, such as uronic acid; (iv) dicarboxylic acids such as malonic acid; (v) tricarboxylic acids, such as propane-1, 2, 3-tricarboxylic acid or aconitic acid; (vi) tetracarboxylic acids, such as 1,2,3, 4-butanetetracarboxylic acid; (vii) pentacarboxylic acids, such as 1,2,3,4, 5-pentanetricarboxylic acid; (viii) polymeric poly (carboxylic acids), such as poly (acrylic acid) or poly (methacrylic acid); (ix) polyaminocarboxylic acids, such as ethylenediaminetetraacetic acid; (x) Amino acids, such as glutamic acid or aspartic acid; (xi) Inorganic acids such as nitric acid, sulfuric acid, hydrogen halides; (xii) Aromatic carboxylic acids such as benzoic acid; (xiii) Acidic pH-sensitive dyes such as, but not limited to, hydroxypyrene and its derivatives (e.g., fluorescein dye, also known as HPTS trisodium salt), phenylpyridyl oxazole and its derivatives (e.g., dextran-conjugated LysosensorTMYellow/blue), aminonaphthalenes and their derivatives (e.g., ANTS), cyanines and their derivatives (e.g., IRDye)TM680RD), triarylmethane dyes (e.g., bromocresol green, bromocresol purple, cresol red, chlorophenol red, phenol red, phenolphthalein, thymol blue, bromothymol blue), azo dyes (e.g., methyl red, chrome black T), nitrophenol dyes (e.g., 2, 4-dinitrophenol), anthraquinone dyes (e.g., alizarin); or (xiv) a combination of at least two thereof; in one embodiment, citric acid is used; in another embodiment, HPTS is used. Use of polymer-conjugated pH-sensitive dyes (e.g. dextran-conjugated Lysosensor)TMYellow/blue, dextran-conjugated fluorescein isothiocyanate, methoxy polyethylene oxide-conjugated fluorescein, dextran-conjugated rhodamine B) may be beneficial in reducing leakage of purified dyes. Any water soluble polymer, such as, but not limited to, dextran, poly (ethylene oxide), poly (vinyl pyrrolidone), or poly (vinyl alcohol)) can then be conjugated to the dye.
Although certain of the above-listed acids may have certain pharmacological activities, at certain dosages, the encapsulated acids used in particular embodiments of the polymersomes of the invention are not intended to exert a direct pharmacological or imaging function, but are used only to generate transmembrane pH gradients and, when the acid is also a pH-sensitive dye, to sense ammonia-related pH changes in the core. The present invention includes the use of any of the above acids, whether or not they also possess certain pharmacological activity. However, according to certain embodiments or aspects of the invention, the acid may also be other than an acid, in addition to any of the acids listed above, which are referred to as antibiotics, anti-cancer agents, anti-hypertensive agents, anti-fungal agents, anti-anxiety agents, anti-inflammatory agents, immunomodulatory agents, anti-viral agents, or lipid lowering agents.
In particular embodiments, the concentration of acid used in the method may vary between 0.1 and 100mM, and the osmolality is from 50 to 700 mOsmol/kg. When citric acid is used, a concentration of about 0.5mM to 50mM, and an osmolality of 150 to 600mOsmol/kg is most preferred. In another embodiment, the osmolality is from 100 to 750 mOsmol/kg. In another embodiment, the osmolality is from 100 to 700 mOsmol/kg. In another embodiment, the osmolality is from 115 to 700 mOsmol/kg. The acid concentration is chosen to avoid affecting the assay sensitivity and also to enable reliable fluorescence or absorbance measurements when the acid used is also a pH sensitive dye.
When the polymersome is hydrated, the concentration of the nucleic acid is such that a pH of 1 to 6.8, in more particular embodiments, a pH of 1 to 6.5, a pH of 2 to 6 results. In a specific embodiment, a pH of about 5.5 is used. In another embodiment, a pH of about 3.0 is used. In another embodiment, a pH of about 2.0 is used.
Application method
The present invention includes methods for quantifying ammonia in various samples using the transmembrane pH gradient polymersomes of the invention. The method can detect ammonia concentrations at least as low as about 0.005mM and at least as high as 8 mM.
In particular embodiments, the precise ammonia concentration in the test sample can be determined by the following method.
According to one method, the concentration of ammonia in a sample can be assessed by contacting the sample with a polymersome of the invention (i.e., containing a pH-sensitive dye) (or with a polymersome-containing composition or a test strip) and measuring at least one pH-dependent spectral characteristic in the polymersome-containing sample, the composition-containing sample, or the sample-containing test strip. Then, in a standard curve of known ammonia concentration, the ammonia concentration in the sample can be deduced by comparing the measured spectral characteristics with the spectral characteristics (i.e., absorbance or fluorescence intensity) at the same pH-dependent wavelength. A standard curve is prepared by first determining the "corresponding reference spectral properties" obtained under the particular conditions used in the assay at each particular ammonia concentration. More specifically, in the core of the same specific transmembrane pH gradient polymersome, the spectral characteristics produced by the same specific pH-sensitive dye at the same specific pH-dependent wavelength are determined in the presence of each specific ammonia concentration outside and inside the polymersome, in the case where the acid concentration at a specific pH in the core is the same as those used to determine samples with unknown ammonia amounts (if any). A "standard curve" is a curve obtained by mathematical curve fitting of a set of corresponding reference spectral characteristics for all tested ammonia concentrations measured under these conditions. The number of test ammonia concentrations used to generate the standard curve is at least 1 (i.e., one concentration may be sufficient if the standard curve is linear within a given range).
As used herein, the term "spectral characteristics" refers to the absorbance or fluorescence intensity in the electromagnetic spectrum from about 10-2000nm, i.e., in the ultraviolet (about 10-390nm), visible (about 390-700nm) and near infrared (NIR, about 700-2000nm) regions of the spectrum.
As used herein, the term "standard curve" is a generic term used to encompass the terms "absorbance standard curve" and "fluorescence standard curve".
Alternatively, the "spectral characteristic ratio" may be determined by normalizing the spectral characteristics of the dyes at a pH-dependent wavelength to the spectral characteristics of the same or different dyes at a pH-dependent wavelength or at another pH-dependent wavelength. If the dye used does not have a pH independent wavelength, a second dye having a pH independent wavelength can be used as a reference to calculate the ratio. The spectral characteristic ratios determined on the sample can then be compared with a universal spectral ratio standard curve resulting from a universal reference spectral characteristic ratio calculated for each ammonia concentration at the same wavelength, and thus the different standard curves obtained for each specific set of assay conditions may no longer be required. Of course, a specific spectral characteristic ratio standard curve ("specific spectral characteristic ratio standard curve") produced from corresponding reference spectral characteristic ratios measured and calculated for a particular assay condition may still be prepared for optimal accuracy.
As used herein, the term "universal reference spectral characteristic ratio" refers to a spectral characteristic ratio in a fluid, in the core of a transmembrane pH-gradient polymersome, produced by a pH-sensitive dye at a pH-dependent wavelength and a pH-independent wavelength (or another pH-dependent wavelength) (at ammonia concentrations both outside and inside the polymersome) at an acid concentration at a given pH. A "universal spectral specific ratio standard curve" is a curve generated by mathematical curve fitting of a set of universal reference spectral characteristic ratios calculated for all tested ammonia concentrations under these conditions. The number of test ammonia concentrations used to generate the standard curve is at least 1 (i.e., one concentration may be sufficient if the standard curve is linear within a given range).
As used herein, the term "spectral characteristic ratio standard curve" is a generic term used to encompass the terms "specific spectral characteristic ratio standard curve" and "generic spectral characteristic ratio standard curve". As used herein, the terms "specific spectral characteristics ratio standard curve" and "universal spectral characteristics ratio standard curve" are used to denote a "specific fluorescence intensity ratio standard curve" and a "specific absorbance ratio standard curve"; and the generic terms "universal fluorescence intensity ratio standard curve" and "universal absorbance ratio standard curve".
As used herein, the terms "pH-dependent wavelength" and "pH-independent wavelength" are used to encompass the terms "pH-dependent fluorescence wavelength" and "pH-dependent absorption wavelength"; and the generic terms "pH-independent fluorescence wavelength" and "pH-independent absorption wavelength".
As used herein, the terms "pH-dependent spectral characteristic" and "pH-independent spectral characteristic" are used to encompass the terms "pH-dependent fluorescence intensity" and "pH-dependent absorbance"; and the general terms "pH-independent fluorescence intensity" and "pH-independent absorbance".
As used herein, the term "spectral characteristic ratio" is a generic term used to encompass the terms "fluorescence intensity ratio" and "absorbance ratio".
More specifically, the aforementioned spectroscopic method may be a fluorescence method or a colorimetric method. Such methods may be further defined as follows.
Fluorescence method
According to one method, the concentration of ammonia in the sample can be deduced by reference to the fluorescence intensity at the same emission and excitation wavelengths in a standard curve of fluorescence intensity for known ammonia concentrations. The fluorescence intensity calibration curve was prepared by first determining the "corresponding reference fluorescence intensity" obtained under the specific conditions used in the assay at each specific ammonia concentration. More specifically, in the core of the same specific transmembrane pH gradient polymersome, the fluorescence generated by the same specific pH-sensitive dye at the same specific pH-dependent wavelength is measured in the presence of each specific ammonia concentration outside and inside the polymersome, in the case where the acid concentration at a specific pH in the core is the same as those used for measuring samples with unknown ammonia amounts (if any). The "standard curve of fluorescence intensity" is a curve obtained by mathematical curve fitting of a set of corresponding reference fluorescence intensities measured for all test ammonia concentrations under these conditions. The number of test ammonia concentrations used to generate the standard curve is at least 1 (i.e., one concentration may be sufficient if the standard curve is linear within a given range).
Alternatively, the "fluorescence intensity ratio" may be determined by normalizing the fluorescence intensity of the dyes at pH-dependent (emission or excitation) wavelengths to the fluorescence intensity of the same or different dyes at pH-independent (absorbance) (emission or excitation) wavelengths. If the dye used does not have a wavelength that is not pH-dependent, a second dye having a wavelength that is not pH-dependent can be used as a reference to calculate the ratio. The fluorescence intensity ratios determined on the samples can then be compared to a universal fluorescence intensity ratio standard curve resulting from a universal reference fluorescence intensity ratio calculated for each ammonia concentration at the same wavelength, and thus the different fluorescence intensity standard curves obtained for each specific set of assay conditions may no longer be required. Of course, a specific fluorescence intensity ratio standard curve ("specific fluorescence intensity ratio standard curve") generated from corresponding reference fluorescence intensity ratios measured and calculated for a specific assay condition can still be prepared for optimal precision.
As used herein, the term "universal reference fluorescence intensity ratio" refers to the ratio of fluorescence intensities produced by a pH sensitive fluorescent dye at pH dependent (emission or excitation) and pH independent (emission or excitation) wavelengths (at ammonia concentrations present both outside and inside the polymersome) at acid concentrations at a defined pH in the core of a transmembrane pH gradient polymersome in a fluid. The "universal fluorescence intensity ratio standard curve" is a curve generated by mathematical curve fitting of a set of universal reference fluorescence intensity ratios calculated for all tested ammonia concentrations under these conditions. The number of test ammonia concentrations used to generate the standard curve is at least 1 (i.e., one concentration may be sufficient if the standard curve is linear within a given range).
As used herein, the term "fluorescence standard curve" is a generic term used to encompass the terms "fluorescence intensity standard curve" and "fluorescence intensity ratio standard curve". As used herein, the term "fluorescence intensity to standard curve" is a generic term used to encompass the terms "specific fluorescence intensity to standard curve" and "generic fluorescence intensity to standard curve".
As used herein, the terms "pH-dependent excitation wavelength" and "pH-independent excitation wavelength" refer to excitation wavelengths whose excitation results in pH-dependent fluorescence intensity and pH-independent fluorescence intensity, respectively, at a particular emission wavelength. As used herein, the terms "pH-dependent emission wavelength" and "pH-independent emission wavelength" refer to emission wavelengths that, if excited at a particular excitation wavelength, exhibit a pH-dependent fluorescence intensity and a pH-independent fluorescence intensity, respectively. As used herein, the term "pH-dependent fluorescence wavelength" refers to a pH-dependent emission wavelength or a pH-dependent excitation wavelength. As used herein, the term "pH-independent fluorescence wavelength" refers to a pH-independent emission wavelength or a pH-independent excitation wavelength.
As used herein, the term "pH-dependent fluorescence intensity" refers to the fluorescence intensity generated at a pH-dependent emission wavelength or a pH-dependent excitation wavelength, or to the fluorescence intensity generated at both a pH-dependent emission wavelength and a pH-dependent excitation wavelength. As used herein, the term "pH-independent fluorescence intensity" refers to the fluorescence intensity generated at a pH-independent emission wavelength and a pH-independent excitation wavelength.
Fluorescence intensity was selected as indicated by the dye supplier, or by recording emission and excitation spectra at different pH values and by identifying pH-dependent and pH-dependent fluorescence wavelengths.
Colorimetric method
According to another method, the ammonia concentration can be deduced by reference to the absorbance at the same ultraviolet or visible or NIR wavelength in an absorption standard curve for a known ammonia concentration. The absorbance standard curve is prepared by first determining the "corresponding reference absorbance" obtained at each particular ammonia concentration under the particular conditions used for the assay. More specifically, in the core of the same specific transmembrane pH gradient polymersome, the absorbance produced by the same specific pH-sensitive dye at the same specific pH-dependent visible light wavelength is determined in the presence of each specific ammonia concentration outside and inside the polymersome, in the case where the acid concentration at a specific pH in the core is the same as those used for determining a sample with an unknown amount of ammonia (if any). The "absorbance standard curve" is a curve obtained by mathematical curve fitting to a set of corresponding reference absorbance values measured for all tested ammonia concentrations under these conditions. The number of test ammonia concentrations used to generate the standard curve is at least 1 (i.e., one concentration may be sufficient if the standard curve is linear within a given range).
As used herein, the term "pH-dependent absorption wavelength" is used to refer to the wavelength at which a dye absorbs light in the ultraviolet (about 10 to 390nm), visible (about 390 to 700nm), or NIR (about 700 to 2000nm) region of the electromagnetic spectrum as a function of the pH of the medium. The absorption wavelength is selected according to the instructions of the dye supplier or by recording the absorption spectra at different pH values and by identifying the pH dependent absorption wavelength and the pH independent absorption wavelength.
As used herein, the terms "pH-dependent absorbance" and "pH-independent absorbance" refer to absorbance at a pH-dependent absorption wavelength and a pH-independent absorption wavelength, respectively.
According to the present invention, conventional laboratory scale or portable spectrophotometers (e.g., standard spectrophotometers for colorimetry or fluorescence spectrophotometers for fluorescence methods) and microplate readers can be used to measure absorbance or fluorescence.
As used herein, a "sample" from which the ammonia (or indirectly phenylalanine) content can be determined according to the present invention can be, but is not limited to, a sample that can contain ammonia or phenylalanine, including biological samples (e.g., body fluid samples), soil samples, wastewater samples, or simple buffers. The sample may be an intrinsic fluid or a fluid formed upon addition of an aqueous solution (e.g., a soil sample). The sample (native fluid or fluid formed after addition of aqueous solution) may be further modified by optional addition of buffers and/or enzymes (e.g., for pH stabilization or adjustment and for ammonia-generating enzymatic reactions, respectively). According to particular embodiments, the sample may comprise or consist of a biological sample (e.g., a bodily fluid). In other embodiments, the sample may further comprise an aqueous solution. In other embodiments, the sample may further comprise at least one buffer. In other embodiments, the sample may further comprise at least one enzyme (e.g., phenylalanine ammonia lyase). In other embodiments, the sample may consist of: (a) biological samples (e.g., bodily fluids); and (b) (i) an aqueous solution; (ii) at least one buffer; (iii) enzymes (e.g., phenylalanine ammonia lyase); or (iv) a combination of at least two of (i) to (iii). It may be unnecessary or useless to remove phenylalanine ammonia lyase from the sample solution prior to mixing with the polymersome.
The incubation time of the polymersomes in the sample may vary depending on the nature of the sample. For biological samples, the optimal incubation time is limited to avoid protein degradation. As shown herein, reliable spectral characteristic (i.e., fluorescence intensity and/or absorbance) readings can be made in biological samples after incubation times as short as 2 minutes and up to 15 minutes (see fig. 2). For non-biological samples (i.e. simple buffers), incubation can be increased while avoiding ammonia evaporation.
As used herein, the term "bodily fluid" refers to any fluid from a vertebrate. In a specific embodiment, it refers to a fluid from a mammal. Without being limited thereto, it includes blood (as in the use of NIR dyes in the fluorescence or absorption method of the invention, or after removal of red blood cells, e.g. via a filter), blood fractions (e.g. serum, plasma), saliva, urine, sweat, semen, peritoneal fluid, fluid from ascites, and cerebrospinal fluid. Certain body fluids may contain ammonia levels or specific amino acid levels that may provide information about the subject (e.g., in terms of the presence or indication of the presence of a disease or disorder in the subject, the effectiveness or non-effectiveness of a therapeutic or prophylactic measure, or the development of side effects caused by administration of a drug). In the examples below, certain body fluids have been diluted with factors selected to fall within the measurement range of a commercial ammonia assay kit for comparison.
Thus, in particular embodiments where the sample being assayed is a body fluid sample, the quantitative methods can be used to diagnose certain diseases or disorders (e.g., ammonia-related diseases or disorders, or diseases characterized by increased levels of particular amino acids (e.g., phenylketonuria)). In more particular embodiments, when the disease or disorder is hyperammonemia, it may be used to diagnose/detect/monitor the disorder in certain hyperammonemia-inducing therapies (e.g., valproic acid therapy). In other embodiments, it can be used to monitor the efficacy of anti-hyperammonemia therapy (e.g., hemodialysis) or anti-phenylketonuria therapy (e.g., low phenylalanine dietary regimens).
As used herein, "ammonia-related disease or disorder" includes hyperammonemia (e.g., caused by impaired liver function or by valproic acid therapy), hepatic encephalopathy, cirrhosis, acute liver failure, acute and chronic liver failure, portal venous shunt, drug-induced hyperammonemia, congenital defects in liver ammonia metabolism (primary hyperammonemia), congenital defects affecting liver ammonia metabolism (secondary hyperammonemia), chronic kidney disease, and ammonia-related decreased fertility. Blood and fractions thereof (serum, plasma) can be used as body fluid samples for most ammonia-related diseases or disorders. Saliva can be used as a body fluid sample for chronic kidney disease and semen can be used as a body fluid sample for ammonia-related fertility reduction.
Certain diseases are characterized by an increased level of amino acids in a body fluid of a subject. For example, phenylalanine levels are elevated in the blood of subjects with phenylketonuria. Phenylketonuria is a congenital metabolic defect that results in decreased levels of phenylalanine hydroxylase (PAH), decreased metabolism of the amino acid phenylalanine, and thus increased blood levels of phenylalanine (vanSpronsen et al, Lancet Diabetes and Endocrinology (Lancet Diabetes Endocrinol). 2017; 5: 743-. Abnormal levels of such amino acids can be indirectly quantified by first incubating the sample with an ammonia-producing enzyme (e.g., phenylalanine ammonia lyase) and then indirectly determining the amino acid level using the ammonia quantification method of the present invention. Phenylalanine ammonia lyase (EC4.3.1.24) is an enzyme that catalyzes the reaction that converts L-phenylalanine to ammonia and trans-cinnamic acid.
As used herein, the term "subject" refers to a subject whose body fluid sample may have ammonia or amino acid levels that can be advantageously quantified by the methods of the invention. It refers to a vertebrate, in a particular embodiment a mammal, and in a more particular embodiment a human. The polymersomes or compositions of the invention may also be used in preclinical research or veterinary applications, and may be used in pets or other animals (e.g., pets such as cats, dogs, horses, etc.; and cattle, fish, pigs, poultry, etc.).
In particular embodiments, the subject has an ammonia-related disease or disorder or phenylketonuria. In another embodiment, the subject is undergoing anti-hyperammonemia therapy (e.g., hemodialysis, liposome-supported peritoneal dialysis) or anti-phenylketonuria therapy (e.g., a low phenylalanine diet regimen).
In another embodiment, the subject is suspected of having or may be suffering from an ammonia-related disease or disorder or phenylketonuria. Without being limited thereto, such subjects include, for example, patients suffering from urea cycle disorders, hepatic encephalopathy, phenylalanine hydroxylase or tetrahydrobiopterin deficiency, as well as patients treated with hyperammonemia-inducing drugs (e.g., L-asparaginase, valproic acid).
Thus, in particular embodiments, the bodily fluid sample may be from any such subject (.
Depending on the type of assay being performed, a "reference ammonia concentration" may be selected from an established standard level of ammonia in a particular body fluid sample, a corresponding ammonia concentration determined at an earlier time (e.g., when the method is used to monitor the effectiveness of an anti-hyperammonemia or anti-phenylketonuria treatment or to induce the effects of an hyperammonemia treatment) in a corresponding sample from the subject; ammonia concentration determined in a corresponding biological fluid of one or more subjects known to be less susceptible to certain diseases or disorders (e.g., ammonia-related diseases or disorders or phenylketonuria) and/or known not to have certain diseases or disorders (e.g., ammonia-related diseases or disorders or phenylketonuria) (e.g., when the method is used to diagnose certain diseases or disorders (e.g., ammonia-related diseases or disorders or phenylketonuria)). In another embodiment, the reference ammonia concentration is an average or median value obtained after determining the ammonia concentration in a plurality of samples (e.g., samples obtained from several healthy subjects or samples obtained from several subjects with certain diseases or disorders (e.g., ammonia-related diseases or disorders or phenylketonuria)).
As used herein, the term "treatment that induces hyperammonemia" refers to a treatment that can result in hyperammonemia or that is a reported side effect. Without being limited thereto, they refer to valproic acid therapy and L-asparaginase therapy (Ando et al, Biopsychology medicine (Biopsichosoc Med); 2017; 11: 19; Strickler et al, leukemia and lymphoma (LeukLymphoma) 2017).
The term "anti-hyperammonemia treatment" as used herein refers to any pharmacological (e.g. sodium phenylbutyrate)
Figure BDA0002357674990000301
Phenylbutyric acid glyceride
Figure BDA0002357674990000302
Sodium phenyl acetate and sodium benzoate
Figure BDA0002357674990000303
Glutamic acid
Figure BDA0002357674990000304
Administration of the non-absorbable disaccharide lactulose rifaximin (e.g. Xifaxan)TM) A spherical carbon adsorbent (AST-120,
Figure BDA0002357674990000305
) And/or administration of transmembrane pH gradient polymersomes (see, e.g., co-pending PCT application PCT/IB2017/054966, matriori and Leroux, supra, filed on 8, 15, 2017)); and/or non-pharmacological (e.g., hemodialysis) therapeutic intervention, aimed at reducing ammonia levels in body fluids. It also relates to any prophylactic measures aimed at preventing the elevation of the level of ammonia in the body fluid (e.g. prophylactic administration of lactulose and/or rifaximin, treatment of spontaneous bacterial peritonitis or gastrointestinal bleeding in HE patients, Vilstrup et al, supra). As used herein, the term "anti-phenylketonuria treatment" refers to any pharmacological (e.g., tetrahydrobiopterin, van Spronsen et al, supra) or non-pharmacological therapeutic intervention (e.g., a low phenylalanine diet regimen, van Spronsen et al, supra) intended to reduce phenylalanine levels in bodily fluids or any prophylactic measure (e.g., a low phenylalanine diet regimen, van Spronsen et al, supra) intended to prevent elevated phenylalanine levels in bodily fluids.
In particular embodiments where the test sample is a soil or wastewater sample, a quantitative method may be used to quantify (e.g., determine the concentration of) ammonia contamination of these substrates (e.g., in the case of wastewater contaminated with ammonia-containing fertilizers or industrial waste).
Composition comprising a metal oxide and a metal oxide
The polymersomes may be mixed in the sample to be tested in different forms (e.g. to determine the ammonia concentration), e.g. may be dispersed in an aqueous medium (e.g. water), possibly together with excipients, such as preservatives, or in their dry form (e.g. deposited on a test strip).
The polymersomes of the invention may be stored as a liquid (e.g., a liquid suspension) or solid (e.g., a powder for reconstitution or deposition on a diagnostic test strip prior to use).
The invention also relates to the use of said polymersomes and/or compositions in the preparation of diagnostic agents.
The compositions of the present invention may contain one or more excipients, including, but not limited to, preservatives (e.g., sodium azide, sorbic acid/sorbate, benzoic acid/benzoate, paraben), antioxidants (e.g., ascorbic acid and its salts, erythorbic acid and its salts), and/or salts. When the polymersomes are in dry form, they may be further combined with cryoprotectants and/or lyoprotectants (sugars, such as trehalose, cane sugar, and sucrose; polyols, such as poly (vinyl pyrrolidone), poly (vinyl alcohol)) and/or extenders (e.g., sugars, cellulose derivatives). The polymersomes can also be incorporated onto a test strip (e.g., a diagnostic test strip). The carrier for such a strip may be, for example, a polymer (paper, film, plastic) or an inorganic support.
Reagent kit
Also within the scope of the invention is a kit comprising (a) at least one type of polymersome, composition and/or strip of the invention; and (b) (i) a solution for hydrating the polymersomes (prior to their use); (ii) a buffer for adjusting the pH (and/or osmolarity) of an external phase or sample to be tested (e.g., a sample, such as a bodily fluid sample (e.g., blood or blood fraction (e.g., serum, plasma)), saliva, urine, tears, semen); (iii) a diluent for diluting a sample to be tested (e.g., a soil sample); (iv) a fluorescence standard curve (e.g., a fluorescence intensity standard curve and/or a fluorescence intensity ratio standard curve) and/or an absorbance standard curve; (v) one or more solutions of known ammonia concentration (standard ammonia solutions); or (vi) a combination of at least two of (i) to (v), and ultimately instructions for its use (e.g., for the quantification of ammonia or for the diagnosis of a particular disease or disorder (e.g., an ammonia-related disease or disorder, such as HE or phenylketonuria).
Number of
The amount of polymersome of the present invention or compositions thereof used in quantitative (e.g., concentration determination) and diagnostic methods will depend on a variety of factors, including the pH of the polymeric vesicle core, the pH-sensitive dye concentration of the polymeric vesicle core, the acid concentration of the polymeric vesicle core, the osmolarity of the polymeric vesicle core, the external phase ammonia concentration, the external phase pH, and the external phase osmolarity. The amount of polymersome of the present invention or a composition thereof will be an amount effective to quantify the amount of ammonia in a sample (e.g., a bodily fluid sample, soil, wastewater, or buffer solution). Without being limited thereto, in one embodiment, the molar concentration of the polymersome in a liquid state is estimated to be in a range of 100nM to 100 mM. In another embodiment, the concentration of polymersomes, expressed as mass concentration of polymer, is 0.01mg/mL to 100 mg/mL.
The present invention includes any combination of polymersomes described herein or compositions comprising the same prepared in the proportions described herein using the solvents described herein, pH-sensitive dyes, and finally acid or acid solutions using the above-described organic and aqueous phase mixing techniques.
The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.
The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted.
Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All subsets of values within the ranges are also incorporated into this specification as if they were individually recited herein.
All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context.
The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed.
No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.
Herein, the term "about" has its ordinary meaning. In embodiments, it may refer to a qualifying value plus or minus 10%. Herein, the term "about" has its ordinary meaning. In embodiments, it may refer to a qualifying value plus or minus 10%. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
The invention is illustrated in further detail by the following non-limiting examples.
Example 1: effect of L-lysine on ammonia quantification based on the fibulo reaction.
And (4) setting an experiment. 0.3mM ammonia (without and with 15mM L-lysine) was mixed at room temperature with fenofibrate reagents (ready-to-use alkaline hypochlorite solution and phenol sodium nitroprusside solution, both from Sigma-Aldrich Chemie GmbH, Switzerland, Buchs) in phosphate buffered saline (1mM potassium dihydrogen phosphate, 3mM sodium hydrogen phosphate, 155mM sodium chloride) at pH 7.4. The absorbance of the solution was measured at 636nm using a spectrophotometer. The presence of L-lysine leads to an underestimation of the ammonia concentration determined by the fibulo reaction. The results are shown in fig. 1 and are expressed as mean and standard deviation (n-3).
Example 2: fluorescence intensity ratio standard curve of transmembrane pH gradient polymersome containing fluorescent yellow dye under different ammonia concentration in phosphate buffer.
And (3) preparing polymersomes. PS-b-PEO polymersomes were prepared using an oil-in-water (o/w) emulsion method. More specifically, 30mg of PS-b-PEO (PS/PEO ratio of about 1.4, PS (2770) -b-PEO (2000), Advanced Polymer materials Co.) was dissolved in 100L of an organic solvent (methylene chloride). The polymer organic solvent solution (polymer-containing organic solvent phase, i.e., oil phase) was added dropwise to 1mL of a pH 5.5 citric acid solution (1mM) containing 10mM of a fluorescent yellow dye (acidic aqueous phase) in an ice bath at an osmolality of 300mOsmol/kg under sonication to form an emulsion having a solvent/aqueous phase ratio of 9% (v/v). The organic solvent was evaporated using a rotary evaporator at 40 ℃ and 700mbar for at least 5 minutes. At this stage of the process, citric acid and a fluorescent dye are present both inside and outside the polymersome. To remove unencapsulated fluorescent dye and exchange the external buffer phase with phosphate buffered saline (PBS, 1mM potassium dihydrogen phosphate, 3mM disodium hydrogen phosphate, 155mM sodium chloride) at pH7.4, 300mOsmol/kg, the polymersome dispersion (exclusion limit 5000g/mol) was purified on a cross-linked dextran gel filtration column. The resulting polymersome encapsulated a citric acid solution at pH 5.5 and a fluorescent dye. The fluorescent dye concentration was quantified using the fluorescence emission intensity at 510nm, measured with a spectrofluorometer, excited at 413 nm.
Quantification of ammonia. Polymersomes containing fluorescent yellow dye (normalized to a fluorescent yellow dye concentration of 0.057 mM) were incubated with PBS solutions of different ammonia concentrations (0-2mM) at pH7.4 at room temperature. At different time points (2.5, 5, 10 and 15 minutes), the fluorescence emission intensity at 510nm excited at 455nm (pH-dependent excitation wavelength) and the fluorescence emission intensity at 510nm excited at 413nm (non-pH-dependent excitation wavelength) were measured using a fluorescence spectrophotometer. The fluorescence intensity ratio was determined by normalizing the former to the fluorescence emission intensity of the latter.
The fluorescence intensity ratio of transmembrane pH gradient PS-b-PEO polymersome containing a fluorescent yellow dye depends on the ammonia concentration in the buffer. The results are shown in fig. 2 and are expressed as mean and standard deviation (n-3).
Example 3: ammonia was quantified in human serum by transmembrane pH-gradient PS-b-PEO polymersomes containing fluorescent yellow dye and a commercial enzymatic ammonia assay.
And (3) preparing polymersomes. Fluorescent transmembrane pH gradient PS-b-PEO polymersomes were prepared and purified as described in example 2, using a modified citric acid concentration of 5mM and a modified external phase (300mOsmol/kg, 50mM phosphate buffer pH 7.4) in a column-based purification method.
Quantification of ammonia. Polymersomes containing fluorescent yellow dye (normalized to a fluorescent yellow dye concentration of 0.016 mM) were incubated with commercially available human serum, 0.1mM ammonia spiked human serum, and PBS solutions containing different ammonia concentrations (0-0.5mM) at room temperature. After 10 minutes, the fluorescence emission intensity at 510nm excited at 455nm (pH-dependent excitation wavelength) and the fluorescence emission intensity at 510nm excited at 413nm (pH-independent excitation wavelength) were measured using a fluorescence spectrophotometer. The fluorescence intensity ratio was determined by normalizing the former to the fluorescence emission intensity of the latter. The ammonia concentration of serum ammonia plus standard serum was determined by comparison to a linear regression curve of fluorescence intensity ratio derived from ammonia standards (fluorescence intensity ratio standard curve). In addition, the same solutions were analyzed using an enzymatic Ammonia kit (Randox Ammonia Assay AM1015, Randox laboratories, inc.) according to the manufacturer's instructions, with the modification that only 30% of the indicated volume was used to enable measurements in 96-well plates with microplate readers.
Similar to the enzyme kit, transmembrane pH-gradient PS-b-PEO polymersome containing a fluorescent yellow dye was able to quantify ammonia in native and spiked human serum. The results are shown in figure 3 and are expressed as mean values and standard deviations (n-3 for polymersome assays and n-8 for enzyme kits).
Example 4: ammonia was quantified in human plasma by transmembrane pH-gradient PS-b-PEO polymersomes containing fluorescent yellow dye and a commercial enzymatic ammonia assay.
And (3) preparing polymersomes. Fluorescent transmembrane pH-gradient PS-b-PEO polymersomes were prepared and purified as described in example 3.
Quantification of ammonia. The ammonia concentration of commercially available human plasma was quantified by fluorescent yellow dye-containing polymersomes and enzyme ammonia kit as described in example 3.
Similar to the enzyme kit, transmembrane pH-gradient PS-b-PEO polymersomes containing fluorescent yellow dye were able to quantify ammonia in native and spiked human plasma. The results are shown in fig. 4 and are expressed as mean and standard deviation (n-3).
Example 5: ammonia in human saliva was quantified by transmembrane pH-gradient PS-b-PEO polymersomes containing fluorescent yellow dye and a commercial enzymatic ammonia assay.
And (3) preparing polymersomes. Fluorescent transmembrane pH-gradient PS-b-PEO polymersomes were prepared and purified as described in example 3.
Quantification of ammonia. The ammonia concentration in commercially available human saliva was quantified by fluorescent yellow dye-containing polymersomes and enzymatic ammonia kit as described in example 3, wherein the modified fluorescent yellow dye concentration was 0.017mM and the modified labeled and unlabeled body fluid formulations (diluted 1:10(v/v) in PBS and labeled with 0.1mM ammonia). Finally, the result is multiplied by the dilution factor.
Similar to the enzyme kit, transmembrane pH-gradient PS-b-PEO polymersomes containing fluorescent yellow dye were able to quantify natural and spiked ammonia in human saliva. The results are shown in fig. 5 and are expressed as mean and standard deviation (n-3).
Example 6: effect of L-lysine on ammonia quantification of transmembrane pH gradient PS-b-PEO polymersomes containing fluorescent yellow dye.
And (3) preparing polymersomes. Fluorescent transmembrane pH-gradient PS-b-PEO polymersomes were prepared and purified as described in example 2.
Quantification of ammonia. The polymersomes containing the fluorescent yellow dye (fluorescent yellow dye normalized to 0.054 mM) were incubated with 0.1mM ammonia-containing PBS solution at pH7.4 in the presence of 0, 1, 5 and 15mM L-lysine and in ammonia standards (0-0.5mM) in PBS at room temperature. After 10 minutes, the fluorescence emission intensity at 510nm excited at 455nm (pH-dependent excitation wavelength) and the fluorescence emission intensity at 510nm excited at 413nm (pH-independent excitation wavelength) were measured using a fluorescence spectrophotometer. The fluorescence intensity ratio was determined by normalizing the former to the fluorescence emission intensity of the latter. The ammonia concentrations of the ammonia solution containing no L-lysine and the L-lysine plus standard ammonia solution were determined by comparison with a linear regression curve (fluorescence intensity ratio standard curve) obtained from the fluorescence intensity ratio of the ammonia standard.
The presence of up to 15mM L-lysine (i.e., 100 times the normal plasma concentration, Aldred et al, J.Autism Dev disease 2003; 33: 93-97) did not affect the ammonia concentration measured by the fluorescent transmembrane pH gradient PS-b-PEO polymersomes. The results are shown in fig. 6 and are expressed as mean and standard deviation (n-3).
Example 7: ammonia in human urine was quantified by transmembrane pH-gradient PS-b-PEO polymersomes containing fluorescent yellow dye and a commercial enzymatic ammonia assay.
And (3) preparing polymersomes. Fluorescent transmembrane pH-gradient PS-b-PEO polymersomes were prepared and purified as described in example 3.
Quantification of ammonia. The ammonia concentration in commercially available human urine was quantified by fluorescent yellow dye-containing polymersomes and an enzymatic ammonia kit as described in example 5, using modified, spiked and untagged body fluid preparations (diluted 1:100(v/v) in PBS and spiked with 0.1mM ammonia). Finally, the result is multiplied by the dilution factor.
Similar to the enzyme kit, transmembrane pH-gradient PS-b-PEO polymersome containing a fluorescent yellow dye was able to quantify ammonia in native and spiked human urine. The results are shown in fig. 7 and are expressed as mean and standard deviation (n-3).
Example 8: polymerization by transmembrane pH gradient PS-b-PEO containing fluorescent yellow dyeVesicle and commercial enzymatic ammonia assay quantify ammonia in human sweat.
And (3) preparing polymersomes. Fluorescent transmembrane pH-gradient PS-b-PEO polymersomes were prepared and purified as described in example 3.
Quantification of ammonia. The ammonia concentration in commercially available human sweat was quantified by fluorescent yellow dye-containing polymersomes and an enzymatic ammonia kit as described in example 3, using modified, labeled and unlabeled body fluid preparations (diluted 1:10(v/v) in PBS and labeled with 0.1mM ammonia). Finally, the result is multiplied by the dilution factor.
Similar to the enzyme kit, transmembrane pH gradient PS-b-PEO polymersomes containing fluorescent yellow dye were able to quantify ammonia in native and spiked human sweat. The results are shown in fig. 8 and are expressed as mean and standard deviation (n-3).
Example 9: ammonia was quantified in human seminal fluid by transmembrane pH gradient PS-b-PEO polymersomes containing fluorescent yellow dye and a commercial enzymatic ammonia assay.
And (3) preparing polymersomes. Fluorescent transmembrane pH-gradient PS-b-PEO polymersomes were prepared and purified as described in example 3.
Quantification of ammonia. The ammonia concentration in commercially available human seminal fluid was quantified by fluorescent yellow dye-containing polymersomes and an enzymatic ammonia kit, as described in example 3, using modified, spiked and untagged body fluid preparations (diluted 1:100(v/v) in PBS and spiked with 0.1mM ammonia). Finally, the result is multiplied by the dilution factor.
Similar to the enzyme kit, transmembrane pH-gradient PS-b-PEO polymersomes containing fluorescent yellow dye were able to quantify ammonia in natural and spiked human semen. The results are shown in fig. 9 and are expressed as mean and standard deviation (n-3).
Example 10: dextran conjugated Lysosensor containing different ammonia concentrations in phosphate bufferTMYellow/blue transmembrane pH gradient polymersome fluorescence intensity ratio standard curve.
And (3) preparing polymersomes. Modified fluorescent dye (dextran conjugated Lysosensor) was used at a concentration of 0.01mM as described in example 3TMYellow/blue, 10000g/mol) and pHFluorescent transmembrane pH gradient PS-b-PEO polymersomes were prepared and purified for a 2.0 modified citric acid solution.
Quantification of ammonia. Dextran conjugate containing Lysosensor was added at room temperatureTMPolymer vesicles of yellow/blue (dextran conjugated Lysosensor standardized to 0.0012 mM)TMYellow/blue concentration) was incubated with PBS solutions of pH7.4 containing different ammonia concentrations (0-0.25 mM). After 10 minutes, the fluorescence emission intensity at 540nm excited at 360nm (pH-dependent emission wavelength) and the fluorescence emission intensity at 485nm excited at 360nm (pH-independent emission wavelength) were measured using a spectrofluorometer. The fluorescence intensity ratio was determined by normalizing the former to the fluorescence emission intensity of the latter.
Dextran-conjugated LysosensorTMThe transmembrane pH gradient of yellow/blue the fluorescence intensity ratio of PS-b-PEO polymersomes depends on the ammonia concentration in the buffer. The results are shown in fig. 10 and are expressed as mean and standard deviation (n-3).
Example 11: fluorescence intensity ratio standard curve of ANTS-containing transmembrane pH-gradient polymersome at different ammonia concentrations in phosphate buffer.
And (3) preparing polymersomes. Fluorescent transmembrane pH-gradient PS-b-PEO Polymer vesicles were prepared and purified as described in example 3 using a modified PS-b-PEO Polymer composition (PS/PEO ratio of about 1.8, PS (3570) -b-PEO (2000), Advanced Polymer Materials) and a modified fluorescent dye (8-aminonaphthalene-1, 3, 6-trisulfonic acid disodium salt, ANTS) and a modified citric acid solution at pH 2.0.
Quantification of ammonia. Polymersomes containing ANTS (normalized to 0.008mM ANTS concentration) were incubated with PBS solutions of pH7.4 containing different ammonia concentrations (0-0.5mM) at room temperature. After 10 minutes, the fluorescence emission intensity at 520nm excited at 368nm (pH-dependent excitation wavelength) and the fluorescence emission intensity at 520nm excited at 308nm (non-pH-dependent excitation wavelength) were measured using a fluorescence spectrophotometer. The fluorescence intensity ratio was determined by normalizing the former to the fluorescence emission intensity of the latter.
The fluorescence intensity ratio of the ANTS-containing transmembrane pH gradient PS-b-PEO polymersomes depends on the ammonia concentration in the buffer. The results are shown in fig. 11 and are expressed as mean and standard deviation (n-3).
Example 12: containing IRDye at different ammonia concentrations in phosphate bufferTM680RD carboxylate standard curve of fluorescence intensity of transmembrane pH gradient polymersome.
Polymer Synthesis of PS (3700) -b-PEO (2000). The synthesis of PS (3700) -b-PEO (2000) was carried out by Atom Transfer Radical Polymerization (ATRP). Monomethyl PEO (2000) was converted to an ATRP macroinitiator by reaction with 2-bromopropionyl bromide in dry Tetrahydrofuran (THF) and further used to polymerize styrene in bulk. Briefly, an ATRP macroinitiator was charged to a flame-dried Schlenk flask along with copper bromide (CuBr) and 4,4 '-dinonyl-2, 2' -bipyridine as catalyst and ligand, respectively. The Schlenk flask was evacuated and refilled with argon to remove oxygen over several cycles. In another flask, styrene was deoxidized by bubbling argon gas thereinto for at least 1 hour, and then charged into a Schlenk flask. The mixture was then heated at 115 ℃ for 16h, and the brown product solution was dissolved in THF, filtered through a basic alumina column, and precipitated twice in hexane. The precipitate was filtered and dried in vacuo. The feed ratio of [ monomer ]/[ initiator ] was 50. The PS/PEO composition was determined by nuclear magnetic resonance spectroscopy.
And (3) preparing polymersomes. Modified fluorescent dye (IRDye) at a concentration of 0.04mM was used with a modified PS-b-PEO polymer composition (PS/PEO ratio of about 1.9, PS (3700) -b-PEO (2000)) as described in example 3TM680RD carboxylate), modified citric acid solution at 20mM concentration and pH 3.0 and modified quantification procedure (PS-b-PEO polymer concentration was prepared and purified by dilution in dimethylformamide at 1:20(v/v) and quantification by measuring absorbance at 271nm using UV spectrophotometer and comparison to PS (3700) -b-PEO (2000) standard curve in dimethylformamide) of fluorescent transmembrane pH gradient.
Quantification of ammonia. At room temperature, will contain IRDyeTM680RD carboxylate polymersomes (PS-b-PEO concentration normalized to 0.73 mg/mL) were incubated with PBS solutions of pH7.4 containing varying ammonia concentrations (0-0.625 mM). After 10 minutes, fluorescence was usedThe spectrophotometer measures the fluorescence emission intensity at 696nm excited at 666 nm.
Containing IRDyeTM680RD carboxylate transmembrane pH gradient PS-b-PEO polymersome fluorescence intensity was dependent on ammonia concentration in buffer. The results are shown in fig. 12 and are expressed as mean and standard deviation (n-4).
Example 13: fluorescence intensity ratio standard curves for transmembrane pH-gradient polymersomes containing fluorescent yellow dye at different ammonia concentrations in phosphate buffer in the absence of other acids in the polymer vesicle core.
Polymer Synthesis of PS (3700) -b-PEO (2000). PS (3700) -b-PEO (2000) was synthesized as described in example 12.
And (3) preparing polymersomes. Fluorescent transmembrane pH-gradient PS-b-PEO polymersomes were prepared and purified as described in example 3 using a 0.9% (m/V) solution of sodium chloride instead of citric acid solution and using a modified PS-b-PEO polymeric composition (PS/PEO ratio of about 1.9, PS (3700) -b-PEO (2000)). Thus, the only acid in the core is the fluorescent dye.
Quantification of ammonia. Ammonia quantification was performed with varying fluorescent yellow dye concentration (0.017mM), incubation time (10 minutes) and ammonia concentration range (0-8mM) as described in example 2.
In the absence of another acid in the core, the fluorescence intensity ratio of the transmembrane pH-gradient PS-b-PEO polymersome containing a fluorescent yellow dye was dependent on the ammonia concentration in the buffer. The results are shown as log-log plots in fig. 13 and are expressed as mean and standard deviation (n-3).
Example 14: fluorescence intensity ratio of transmembrane pH-gradient polymersome containing fluorescent yellow dye with modified PS-b-PEO polymer composition at different ammonia concentrations in phosphate buffer.
Polymer Synthesis of PS (2400) -b-PEO (2000). The PS (2400) -b-PEO (2000) synthesis was performed by ATRP. Monomethyl PEO (2000) was converted to an ATRP macroinitiator by reaction with 2-bromoisobutyryl bromide in dry THF and further used to bulk polymerize styrene. Briefly, an ATRP macroinitiator was charged to a flame-dried Schlenk flask along with CuBr and 4,4 '-dinonyl-2, 2' -bipyridine as catalyst and ligand, respectively. The Schlenk flask was evacuated and refilled with argon to remove oxygen over several cycles. In another flask, styrene was deoxidized by bubbling argon gas thereinto for at least 1 hour, and then charged into a Schlenk flask. The mixture was then heated at 115 ℃ for 3 hours, and the brown product solution was dissolved in THF, filtered through a basic alumina column and precipitated twice in hexane. The precipitate was filtered and dried in vacuo. The feed ratio of [ monomer ]/[ initiator ] was 28. The PS/PEO composition was determined by nuclear magnetic resonance spectroscopy.
And (3) preparing polymersomes. Fluorescent transmembrane pH-gradient PS-b-PEO polymersomes were prepared and purified as described in example 3, using a modified PS-b-PEO polymer composition (PS/PEO ratio of about 1.2, PS (2400) -b-PEO (2000), and PS/PEO ratio of about 3.0, PS (6000) -b-PEO (2000), advanced polymer Materials, inc.) and the amount of modified polymer (10mg) for PS (6000) -b-PEO (2000).
Quantification of ammonia. The polymersomes containing the fluorescent yellow dye (standardized to a fluorescent yellow dye concentration of 0.007mM for PS (2400) -b-PEO (2000) and 0.002mM for PS (6000) -b-PEO (2000)) were incubated at room temperature with PBS solutions containing different ammonia concentrations (0-0.5mM) and PBS solutions containing 0.2mM ammonia, all at pH 7.4. After 10 minutes, the fluorescence emission intensity at 510nm excited at 455nm (pH-dependent excitation wavelength) and the fluorescence emission intensity at 510nm excited at 413nm (pH-independent excitation wavelength) were measured using a fluorescence spectrophotometer. The fluorescence intensity ratio was determined by normalizing the former to the fluorescence emission intensity of the latter. The ammonia concentration of 0.2mM ammonia-containing solution in PBS was determined by comparison with a linear regression curve (fluorescence intensity ratio standard curve) derived from the fluorescence intensity ratio of ammonia standards.
Transmembrane pH gradient PS-b-PEO polymersomes containing fluorescent yellow dye (PS/PEO ratio of about 1.2 and 3.0) were able to quantify ammonia in phosphate buffer. The results are shown in fig. 14 and are expressed as mean and standard deviation (n-3).
Example 15: determination of phenylalanine by transmembrane pH gradient PS-b-PEO polymersome containing fluorescent yellow dye in bufferAmount of the compound (A).
Polymer Synthesis of PS (3700) -b-PEO (2000). PS (3700) -b-PEO (2000) was synthesized as described in example 12.
And (3) preparing polymersomes. Fluorescent transmembrane pH-gradient PS-b-PEO polymersomes were prepared and purified as described in example 3 with a modified PS-b-PEO polymeric composition (PS/PEO ratio of about 1.9, PS (3700) -b-PEO (2000)).
Quantification of phenylalanine. Commercial phenylalanine ammonia lyase from Rhodotorula glutinis (Rhodotorula glutinis) (EC number 4.3.1.5, grade I, activity: 0.8-2.0 units/mg protein (1 unit per minute converts 0.001mmol phenylalanine at pH8.5, 30 ℃) using centrifugation filtration (30 kDa cut-off), Sigma-Aldrich Chemie GmbH) was purified 8 times at 15000x g for 2 minutes. Phenylalanine ammonia lyase (0.013mg/mL) was incubated with different phenylalanine solutions (0-1.2mM, used as standard curve) and phenylalanine test solutions (nominal concentration 0.625mM) in 5mM tris (hydroxymethyl) aminomethane (300mOsmol/kg, pH 8.5) at 30 ℃ for 15 min. Subsequently, aliquots of these solutions were incubated with fluorescent yellow dye-containing polymersomes (normalized to a pyronin concentration of 0.017mM) in 50mM phosphate buffer (300mOsmol/kg, pH7.4 (final pH of the dispersion is 7.4)) at room temperature. After 10 minutes, the fluorescence emission intensity at 510nm excited at 455nm (pH-dependent excitation wavelength) and the fluorescence emission intensity at 510nm excited at 413nm (pH-independent excitation wavelength) were measured using a fluorescence spectrophotometer. The fluorescence intensity ratio was determined by normalizing the former to the fluorescence emission intensity of the latter. The concentration of the phenylalanine test solution was determined to be 0.631 ± 0.022mM (mean and standard deviation, n ═ 3) using the phenylalanine fluorescence intensity ratio standard curve.
Transmembrane pH gradient PS-b-PEO polymersome containing a fluorescent yellow dye can measure the phenylalanine concentration in the buffer after incubation with phenylalanine ammonia lyase.
Example 16: absorbance ratio standard curves for transmembrane pH gradient polymersome containing fluorescent yellow dye at different ammonia concentrations in phosphate buffer.
Polymer Synthesis of PS (3700) -b-PEO (2000). PS (3700) -b-PEO (2000) was synthesized as described in example 12.
And (3) preparing polymersomes. Transmembrane pH-gradient PS-b-PEO polymersomes were prepared and purified as described in example 3 with a modified PS-b-PEO polymeric composition (PS/PEO ratio of about 1.9, PS (3700) -b-PEO (2000)).
Quantification of ammonia. Polymersomes containing fluorescent yellow dye (normalized to a fluorescent yellow dye concentration of 0.01mM) were incubated with PBS solutions of different ammonia concentrations (0-0.5mM) at pH7.4 at room temperature. After 10 minutes, the absorbance was measured at pH-dependent absorption wavelengths of 450nm and 405nm using a spectrophotometer. The absorbance ratio was determined by normalizing the former to the latter absorbance.
The absorbance ratio of the transmembrane pH gradient PPS-b-PEO polymersome containing the fluorescent yellow dye depends on the ammonia concentration in the buffer. The results are shown in fig. 15 and are expressed as mean and standard deviation (n-3).
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Claims (43)

1. A polymersome comprising (a) a membrane comprising a block copolymer of poly (styrene) (PS) and poly (ethylene oxide) (PEO), wherein the PS/PEO molecular weight ratio is higher than 1.0 and lower than 4.0; and (b) a core encapsulating the acid and at least one pH-sensitive dye.
2. The polymersome of claim 1, wherein the block copolymer is a diblock copolymer.
3. Polymersomes according to claim 1 or 2, wherein the concentration of the acid is such as to yield a pH between 1 and 6.5, 2 and 6, 2 and 5.5 or 3 and 5.5 when the polymersomes are hydrated.
4. The polymersome of any one of claims 1 to 3, wherein the acid is in an acidic aqueous solution.
5. The polymersome of claim 4, wherein the pH in the acidic aqueous solution is between 1 and 6.5, 2 and 5.5, or 3 and 5.5.
6. The polymersome of any one of claims 1 to 5, wherein the at least one pH-sensitive dye comprises (i) hydroxypyrene; (ii) phenyl pyridyl oxazole; (iii) an aminonaphthalene; (iv) a cyanine; or (v) any pH-sensitive fluorescent derivative of any one of (i) to (iv).
7. The polymersome of claim 6, wherein the pH-sensitive dye comprises 8-hydroxypyrene-1, 3, 6-trisulfonate (HPTS), dextran-conjugated LysosensorTMYellow/blue, 8-aminonaphthalene-1, 3, 6-trisulfonate (ANTS) or IRDyeTM680RD carboxylate.
8. The polymersome of any one of claims 1 to 7, wherein the acid and the at least one pH-sensitive dye are different molecules.
9. The polymersome of any one of claims 1 to 8, wherein the acid is a hydroxy acid, most preferably citric acid.
10. The polymersome of any one of claims 1 to 7, wherein the acid and the at least one pH-sensitive dye are the same molecule.
11. Polymersomes according to any one of claims 1 to 10, prepared by a process comprising mixing an organic solvent containing the copolymer with an aqueous phase containing the acid and at least one pH-sensitive dye.
12. The polymersome of claim 11, wherein the organic solvent is water insoluble or partially water soluble.
13. The polymersome of any one of claims 1 to 12, wherein the pH-sensitive dye is a pH-sensitive fluorescent dye.
14. The polymersome of any one of claims 1 to 12, wherein the pH-sensitive dye is a pH-sensitive absorbing dye.
15. A method of preparing the polymersome of any one of claims 1 to 14, comprising:
(a) dissolving the block copolymer of PS and PEO in an organic solvent, preferably a water-insoluble or partially water-soluble organic solvent, to form a copolymer-containing organic phase;
(b) mixing the copolymer-containing organic solvent phase with an aqueous phase containing an acid and at least one pH-sensitive dye to form the polymersome; and
(c) removing the at least one pH sensitive dye and the organic solvent that are not encapsulated.
16. The method of claim 15, wherein the aqueous phase comprises 0.2 to 100mM of acid.
17. A polymersome prepared by the method of claim 15 or 16.
18. The polymersome of any one of claims 1 to 14 and 17, the core of the polymersome further encapsulating ammonia.
19. A composition comprising the polymersome of any one of claims 1 to 14 and 17 and at least one excipient.
20. The composition of claim 19, wherein the at least one excipient comprises a preservative, a cryoprotectant, a lyoprotectant, an antioxidant, or a combination of at least two thereof.
21. The composition of claim 19 or 20, wherein the composition is in liquid or solid form.
22. A test strip comprising the composition in solid form of claim 19 or 20.
23. The polymersome according to any one of claims 1 to 14 and 17, or the composition according to any one of claims 19 to 21, or the test strip according to claim 22, for quantifying ammonia in a fluid sample.
24. The polymersome, the composition or the test strip for use according to claim 23, wherein the sample comprises a bodily fluid from a subject.
25. A polymersome, composition or strip for use according to claim 24, wherein the sample further comprises a buffer.
26. A polymersome, composition or strip for use according to claim 24, wherein the subject (i) has an ammonia-related disease or disorder or phenylketonuria; (ii) suspected or likely to suffer from an ammonia-related disease or disorder or phenylketonuria; or (iii) is undergoing treatment for anti-hyperammonemia or anti-phenylketonuria.
27. A method of determining the concentration of ammonia in a sample using the polymersome of any one of claims 1 to 14 and 17, the composition of any one of claims 19 to 21, or the test strip of claim 22, comprising:
(a) contacting the polymersome, composition, or strip with the sample;
(b) determining at least one pH-dependent spectral characteristic in the sample containing polymeric vesicles or composition or the test strip containing the sample; and
(c) determining the concentration of ammonia in the sample using the at least one pH-dependent spectral characteristic by reference to a standard curve.
28. The method of claim 27, wherein the pH-dependent spectral characteristic is pH-dependent absorbance, the pH-sensitive dye is a pH-dependent absorption dye, and the standard curve is an absorbance standard curve.
29. The method of claim 27, wherein the pH-dependent spectral characteristic is pH-dependent fluorescence intensity, the pH-sensitive dye is a pH-sensitive fluorescent dye, and the standard curve is a fluorescent standard curve.
30. The method of claim 27, wherein (b) further comprises measuring at least one pH independent spectral characteristic or at least one additional pH dependent spectral characteristic in the sample containing the polymer vesicle or composition or the sample containing strip to calculate at least one spectral characteristic ratio, and wherein (c) the ammonia concentration in the sample containing the polymer vesicle or composition or the sample containing strip is determined by reference to a spectral characteristic ratio standard curve using the at least one pH dependent spectral characteristic ratio.
31. The method of claim 30, wherein the at least one pH-dependent spectral characteristic and the at least one pH-independent spectral characteristic are produced by the same pH-sensitive dye.
32. The method of claim 30 or 31, wherein the spectral characteristic is absorbance and the pH-sensitive dye is a pH-sensitive absorbing dye.
33. The method of claim 30 or 31, wherein the spectral characteristic is fluorescence and the pH-sensitive dye is a pH-sensitive fluorescent dye.
34. The method of any one of claims 27 to 33, wherein the pH in the polymeric vesicle core is between 2 and 6.5.
35. The method of any one of claims 27 to 34, wherein the at least one pH-sensitive dye comprises hydroxypyrene or one of its derivatives.
36. The method of claim 35, wherein the at least one pH-sensitive dye comprises 8-hydroxypyrene-1, 3, 6-trisulfonate (HPTS).
37. The method of any one of claims 27 to 34, wherein the at least one pH-sensitive dye comprises pyridylphenyloxazole or one of its derivatives; aminonaphthalene or one of its derivatives; or a cyanine or one of its derivatives.
38. The method of claim 37, wherein the at least one pH-sensitive dye comprises a dextran-conjugated LysosensorTMYellow/blue, ANTS or IRDyeTM680RD carboxylate.
39. The method of any one of claims 27 to 38, wherein the sample comprises a sample of bodily fluid from a subject.
40. The method of claim 39, wherein the bodily fluid is a blood or blood fraction sample, a saliva sample, or a semen sample.
41. The method of claim 40, wherein the body fluid has been pretreated with phenylalanine ammonia lyase.
42. The method of claim 40 or 41, which is used (i) to diagnose ammonia-related disease or disorder or phenylketonuria in the subject, wherein an ammonia concentration in the sample that is higher than a reference ammonia concentration is indicative of the subject having ammonia-related disease or disorder or phenylketonuria; or for (ii) monitoring the effectiveness of an anti-hyperammonemia or anti-phenylketonuria treatment, wherein an ammonia concentration in the sample that is lower than a reference ammonia concentration indicates that the anti-hyperammonemia or anti-phenylketonuria treatment is effective.
43. A kit for determining the concentration of ammonia in a sample comprising (a) a polymersome according to any one of claims 1 to 14 and 17, a composition according to any one of claims 19 to 21 or a test strip according to claim 22, and (b) (i) a solution for hydrating the polymersome; (ii) a buffer for adjusting the outer phase of the polymersome and/or the pH of the sample to be tested; (iii) a diluent for diluting a sample to be tested; (iv) a fluorescence standard curve and/or an absorbance standard curve; (v) one or more solutions with known ammonia concentrations; or (vi) a combination of at least two of (i) to (v).
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